WO2022046954A1 - Appl cells improved placental-derived adherent cells and methods of their use - Google Patents

Appl cells improved placental-derived adherent cells and methods of their use Download PDF

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Publication number
WO2022046954A1
WO2022046954A1 PCT/US2021/047626 US2021047626W WO2022046954A1 WO 2022046954 A1 WO2022046954 A1 WO 2022046954A1 US 2021047626 W US2021047626 W US 2021047626W WO 2022046954 A1 WO2022046954 A1 WO 2022046954A1
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Prior art keywords
cells
placental
isolated
population
specific embodiment
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PCT/US2021/047626
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French (fr)
Inventor
Qian Ye
Xiaokui Zhang
Robert J. Hariri
Chenfei HUANG
Kathy KARASIEWICZ-MENDEZ
Shuyang He
Joseph Gleason
Lin KANG
John HARIRI
Chloe HOLLAND
Michael CURTO
Timothy WILK
Navjot SHAH
Valentina ROUSSEVA
Bhavani Stout
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Celularity Inc.
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Publication of WO2022046954A1 publication Critical patent/WO2022046954A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • APPL exitibit a phenotype of CD34 -, CD10 +, CD105 +, and CD200 GE2 secretion is used as potency assay for APPL in Diabetic Foot Ulcer (DFU) with and without Peripheral Arterial Disease (PAD) and Diabetic Peripheral Neuropathy (DPN), Like their bone marrow counterparts, APPL possess potent immune suppressive properties and exert their effects on T lymphocytes through a multitude of mechanisms that include cell contact mediated interactions and through the modulation of secreted soluble factors. APPL have been shown to inhibit both the proliferation and cytokine production of T lymphocytes* as well as modulate T cell differentiation!,
  • TF Tissue factor
  • CD142 Tissue factor
  • TF also called CD142
  • TF plays an essential role in coagulation and hemostasis.
  • the TF :FV!la complex is the key initiator of foe coagulation protease cascade and activates both FIX to PlXa and FX to FXa. this leads to the formation of low amounts of thrombin * which activates the tofactots PV and FVHL2,3
  • Different MSG products display vary ing levels of highly procoagalamtissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (1BM1R).
  • MSCs derived from perinatal tissue (PT) has higher expression of TF than those from adipose tissue (AT) and bone marrow (DM).
  • PT perinatal tissue
  • AT adipose tissue
  • DM bone marrow
  • PDA-001 Human placenta-deri ved cells
  • the present invention provides a population of placental stem cells which are
  • the population of placental stem cells ate additionally negative for one or more markers selected from the group consisting of CD117, CD45, and CD133, In some embodiments, rite population of placental stent cells am additionally negative for CD117. Some embodiments, the population of placental stent cells am additionally Negative for CD45. In some embodiments, depopulation Of placental stem cells are additionally negative for CD133.
  • the population of placental stem cells are additionally positive for One of mom markers selected from the group consisting of CD44, CD90, OcH, and HLA-G.
  • the population of placental stera cells are additionally positive for CD44.
  • the population of placental stem cells are additionally positive for CD90.
  • tire population of placental stem cells are additionally positive fat Oct-4.
  • the population of placental stem cells are additionally positive for HLA-G.
  • the population of placental stem cells are genetically modified.
  • the genetic modification is a tissue factor (CDl 42, F3 gene) knockout-
  • the genetic modification is a CRISPR tissue factor (CD142, F3 gene) knockout.
  • the cells induce increased anti-inflammatory macrophage activity relative to CD142+ PDAC cells, in some embodiments, the increased anti-mflammatory activity comprises increased expression of CDSO on M2 macrophage. In some embodiments, the cells induce increased pro-inflammatory maerophageactivity relative to CDl 42+ PDAC cells. In some embodiments, the increased anti-inflammatory activity comprises increased expression of CD206 on Ml macrophages.
  • the cells have increased antt-inflammatory properties relative' to CD142+ PDAC cells, in some embodiments, the cells express increased an increased marker of activity under inflammatory conditions relative to CD 142+ PDAC cells. In some embodiments, the increased marker of activity is PGE2. In some embodiments, the inflammatory condition is IL-lb stimulation.
  • the present invention also provides a pharmaceutical composition comprising the population of placenta) stem celts of tire invention and a pharmaceutically acceptable earner.
  • 00014 Hie present invention also provides a method of treating a disease or condition in a subject in need thereof, the method comprismg the step of administering to the subject a therapeutically effective amount of the population of placental stem cells of the invention or the pharmaceutical composition of the invention, so as to thereby treat the subject
  • the subject is a mammal.
  • the subject is a human, administration is intravenous.
  • the administration is local.
  • the administration is intramuscular;
  • the subject has an autoimmune disease.
  • the autoimmune disease is multiple sclerosis.
  • the subject has an inflammatory bowel disease.
  • the inflammatory bowel disease is Crohn's disease;
  • the present invention also provides & method of suppressing an tmmune response comprising contacting a plurality of immune cells with a population of placental stem ceils of the invention for & time sufficient fear said placental stem cells to delectably suppress an immune response.
  • the plurality of immune cells are T cells or NK (natural killer) cells;
  • the T cells are CD4+ T cells.
  • the T cells are CD84 T cells, til some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some embodimems, the contacting is performed in a mammalian subject In some embodiments, the mammalian subject is a human subject.
  • FIGS. 1A - 1B show sgRNAs-mediated TFKO in 981 and 995 at different seeding density by FACS and TIDE.
  • FIGS.2A - 2B show reproducibility of TFKO by sgRNA B or C in 981, 974, and
  • FIGS. 3A - 3B show that IHO reduced TF activity in both whole cells and ceil
  • FIGS.4A - 4C show that TF expression and activity was maintained as APFL- TFKO expanded to p6.
  • FIG.5 sbows that TFKO did not cbange protiferation of APPL tines.
  • FIG.6 shows that TFKO did not alter nominal phenotype of APPL.
  • FIG. shows PGE2 secretion of APPL lines,
  • FIGS. SA - 8B show TFKO by sgRNA-B in AB and AL.
  • FIG, 9 shows TFKQ confirmation by TIDE assay
  • FIG, 10 shows the viability of AE arid AL
  • FIG. 11 shows TF activity of 981, 974, AE, and AL at late passage.
  • FIG. 12 shows the nominal phenotypes of AE and AL
  • FIGS. 13 ⁇ - 13B show that suppression of T-cell proliferation was maintained in
  • FIG. 14 shows M1/M2 macrophage polirization induced by APPL-CTR/TFKO.
  • FIG, 0 shows TF expression and phenotype analysis of 4 ⁇ study cell lines.
  • FIG. i 6 shows PGE2 secretion pf APPL*control and -TFKO cells.
  • FIGS. 17A - 17J show secretome analysis of AE, AL, 981 , and 974 cells.
  • FIG. 18 shows the results of an BAB study of AE-control and -TFKO.
  • FIGS. 19A- 19B show cytokine characterization of APPL-derived exosomes.
  • FIG. 20 shows off-target analysis of TFKO in APPL cells by RNA-seq.
  • SH2 refers to an antibody that binds an epitope on the marker CD 105.
  • cells that are referred tons SH2+ are CD105+.
  • SH3 and SH4 refer to antibodies that bind epitopes present on the marker CD73, Thus, cells that are referred to as SH3+ and/or SH4+ are CD73+.
  • immunomodulation and “immunomodulatory” mean causing, or having the capacity to cause, a detectable change in an immune response, and the ability to cause a detectable change m an immune response;
  • immunosuppression and “immunosuppressive” mean causing, or having the capacity to cause, a detectable redaction m an immune response, and the ability to cause a detectable suppression of an immune response.
  • derived means isolated from or otherwise purified
  • placental derived adherent cells are isolated from placenta
  • the term "derived” encompasses cells that are cultured from cells isolated directly from a tissue, eg., the placenta, and cells cultured or expanded from primary isolates.
  • a cell is “isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of toe cells, eg., non-stem cells, with which toe stem cell is naturally associated, or stem cells displaying a di fferent marker profile, ate removed from the stem bell. eg,, during collection and/or Culture of the stem cell.
  • the term " population of isolated cells” means a population of cells that is substantiaily separated from other cells of a tissue, eg., placenta, from which the population of cells is derived.
  • the term “placental cell” refers to a stem cett dr progenitor cell that is isolated from a mammalian placenta, eg. , as described in Section 4, 1 , below, or cultured from cells isolated from a mammalian placenta, either as a primary isolate or a cultured cell, regardless of the number of passages after a primary culture, to certain embodiments, the term “placental cells, “ as osed herein does not, however, refer to frophoblasts, cytotrophoblasts, synchiottdphoblasts, angioblasts, hemangioblasts, embryonic germ cells, embryonic stem cells, cells obtained from an inner cell mass of a blastocyst, or cells obtained from a gonadal ridge of a late embryo, an embryonic germ cell,
  • a placental cell is “positive” for a: particular marker wheat that marker is detectable above background Detection of a particular marker can, for example, be accomplished either by use of antibodies, or by oligonucleotide probes or printers based On the sequence of toe gene ormRNA encoding toe marker.
  • a placental cell is positive for, eg. , CD73 because CD73 is detectable on placental cells in an amount deiectebly greater than background (in comparison to, e.g., an isotype control).
  • a cell is also positive for a marker when that mark» can be used to distinguish the cell from at least one other cell type, or can be used to select or isolate toe cell when present or expressed by the cell, in the context of, eg., antibody-mediated detection, “positive,” as an indication a particular cell surface marker is present, means that the marker is detectable using an antibody, e.g., a fluorescently-labeled antibody, Specific for thatmarker; “positive” also refers to a cell exhibiting toe matker m an amount that produces a Signal, e.g. , in a cytometer, that is delectably above background.
  • a cell is “CD200* where the cell is delectably labeled with aft antibody specific to CD200, and the signal from the antibody is ttetectably higher than that of a control (e g., badtgrotittd or an isotype control).
  • a control e g., badtgrotittd or an isotype control.
  • negative * * in the samo context means that the cell surface marker is hot detectable «sing an antibody specific for that marker compared a control (eg., background or an isotype control).
  • a cell Is “CD34 ” where the cell is not reproducib!y delectably labeled with an antibody specific to CD34 to a greater degree than a control background or an isotype control). Markers not detected.
  • a cell or population Of celis canbe determined to be OCT-4' i f the amount of OCT-4 RNA detected in RNA from the cell or palliation of cells is delectably greater than background as determined, e.g., by a method of detecting RNA such as RT-PCR, slot blots, etc.
  • cluster of differentiation (“CD”) markers are detected using antibodies.
  • OCT-4 is determined to be present, and a cell is "OCT-4" if OCT-4 is detectable using RT-PCR.
  • the terms “subject,” “patient,” and “individual” may be used interchangeably to refer to a mammal being treated with a method Of treatment described herein, In a specific embodiment die subject to be treated b a human.
  • DPN diabetic peripheral neuropathy
  • a therapeutically effective amount of tissue culture plastic-adherent placental cells e.g., placental stem cells, e.g., CD34 , CD 10' , CD 105' , CD200' placental stem cells.
  • placental stem cells e.g., CD34 , CD 10' , CD 105' , CD200' placental stem cells.
  • said placental cells are formulated as a pharmaceutical composition.
  • a subject with DPN treated m accordance with the methods provided herein has type I diabetes.
  • a subject with DPN treated in accordance with the methods provided herein has type II diabetes.
  • a subject treated m accordance with the methods provided herein has DPN in one or more of die arms, hands, legs, or feet .
  • a subject treated in accordance with the methods provided herein has DPN in each of the antis, hands, legs, and feet.
  • a subject treated in accordance with the methods provided herein has DPN and also has a condition that causes a disruption in the flow of blood in the subject’s eripheral vasculature, in a specific embodiment, die sobject hasperipheral arterial disease (PAD).
  • PAD die sobject hasperipheral arterial disease
  • the subject bas peripheral vascular disease. lit cermin embodiments, the methods provided hereto result to a detectable improvement of one or more symptoms of DPH to a subject treated to accordance with die methods provided herein.
  • Exemplary symptoms ofDPN include, without limitation, numbness or reduced ability to feel pain or temperature changes, a tingling or burning sensation: to the limbs, sharp pains or cramps, increased sensitivity to touch, muscle weakness, loss of reflexes (e.g., to the ankle), lost of balance and/or coordination, foot problems (such as ulcers, infections, deformities, and bone and joint pain),
  • the methods provided hereto comprise admtoistering placental stem cells (e.g., a pharmaceutical composition comprising placental stein ceils) to a subject having DPM in an amount and for a time sufficient for detectable improvement to one or more indicia of impro vement.
  • said indicia of improvement is a change in the epidermal nerve fiber density following treatment, as compared to haseline.
  • Epidermal nerve fiber density is a measurement used to assess toe extent of peripheral diabetic neuropathy*
  • To assess epidermal nerve fiber density the number of nerve fibers in a skin biopsy is determined. An increase to toe number/density of nerve fibers is indicative of improving neuropathy.
  • toe methods provided herein comprise administering placental stem cells (e.g., a ph&mtaceutical composition comprising placental stem cells) to a subject havtog DPN in an amount and for a time sufficient for detectable improvement to totality of life of the subject asassessed by, e.g., (i)a364tem Short Form Itoalth Survey (SF-36) (see, e.g., Waro et al., Medical Care 30(6):473483); (ii) the Diabetic peripheral neuropathy Scale Shott Fonn (DFS-SF), which measures toe impact of diabetic peripheral neuropathy tot quality of life (see, e.g..).
  • placental stem cells e.g., a ph&mtaceutical composition comprising placental stem cells
  • toe placental cells e.g., a pharmaceutical composition comprising placental stent ceils
  • placental cells e.g., a pharmaceutical composition comprising placental stem cells
  • the ptacental cells are administered intramuscularly .
  • the placental cells e.g., a pharmaceutical composition comprising placental stem cells
  • the placental cells are administered intramuscularly in the area of the DPN (e.g, in one or more of die legs; feet, arms, or hands)
  • the placental cells are administered intramuscularly adjacent to the area of the DPN.
  • the placental ceHs te.g., a pharmaceutical composition comprising placental stem cells are administered below the knee and above me ankle of a subject that has been diagnosed with DPN.
  • the placental celts e.g., a pharmaceutical composition comprising placental stem cells
  • the placental cells are administered subcutaneously.
  • the placental cells are administered locally.
  • die placental cells e.g., a pharmaceutical composition comprising placental stem cells
  • the methods of treatment of DPN described herein comprise adtmnistration of about 1 x 10 3 , 3 x 10 3 , 5 x 10 3 , 1 x 10 4 , 3 x 10 4 5 x 10 4 , 1 x 10 5 , 3 x 10 5 , 5 x 10 5 , 1 x 106, 3 x M 5 x W, 1 ⁇ tO 7 , 3 k 10 T , $ x 10 7 1 x 10*, 3 x lO*, 5 x 10* 1 x 10 ⁇ 5 X Id*, or 1 x 10 to placental cells (eg., as part of a pharmaceutical composition comprising placental stem cells), to certain embodiments, the methods of treatment of DPN described hereto comprise administration of about 1 x 10 to placental cells (eg., as part of a pharmaceutical composition comprising placental stem cells), to certain embodiment
  • the methods of treatment of DPN described herein comprise administration of about 3 x 10 6 placental cells. In another specific embodiment the methods of treatment of D ⁇ described herein comprise administration of about 1 x 10 7 placental pells. In another specific embodiment, the methods of treatment of DPN described herein comprise administration of about 3 x 10 7 placental cells
  • the isolated placental cells sometimes referred to herein as PDACs useful in the methods of treatment of DPN provided herein are obtainable from a placenta or part thereof adhere to a tissue culture substrate and have characteristics of multipoint cells or stem cells, but are not trophoblasts, in certain embodiments, the isolated placental cells useful m the methods disclosed herein have the capacity to differentiate into non-placental cell types.
  • the isolated placental ceils useful in the methods disclosed herein can be either fetal or matemal tn origin (that is, can have tire genotype of either the fetus or mother, respectively).
  • Be isolated placental cells and populations of isolated placental cells ate fetal in origin.
  • the phrase “fetal origin” or “hon-matemal in origin* indicates that Be isolated placenta cells or populations of isolated placental cells are obtained from Be umbilical cor dor placental structures associated witit the ferns, l m, that have rite fetal genotype.
  • the phrase “maternal in origin” indicates that the cells or populations of cells are obtained from a placental structures associated with the mother , eg, which have the maternal genotype.
  • Isolated placental cells eg, PDACs, or populations of cells comprising the isolated placental cells
  • Bat are solely fetal dr maternal in origin, or dan comprise a mixed population of isolated placental cells of both fetal and maternal origin;
  • the isolated placental celK and populations of cells comprising the isolated placental cells can be identified and selected by Be morphological, marker, and culture characteristics discussed below, hi certain embodiments, any of Be placental cells, eg, placental stem cells or placental multipotent cells described herein, are autologous to 8 recipient, eg., an individual who has a DPN.
  • any of the placental cells eg., placental stem cells or placental multipotent celts described herein, am heterologous to a recipient, eg., an individual who has a DPN. 4.1.1 0 058
  • the isolated placental cells described herein (PDACs) when culturedin primary caUures or in cCti culture, adhere to the tissue ail tore substrate, &g., tissue culture container surface (eg.; tissue culture plastic), or to a tissue cufctoe surface coated with extracellular matrix or ligands such as laminin, collagen (eg.
  • the isolated placental cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmie processes extending from the central cell body.
  • the cells are, however, morphologically distinguishable from fibroblasts cultured under the same conditions, as the isolated placental cells exhibit a greater number of such processes than do fibroblasts. Morphologically, isolated placental cells are also distinguishable from hematopoietic stent cells, Which generally assume a more rounded, or cobblestone, morphOTOgy in culture.
  • the isolated placental cel ts useful when cultured in a growth medium, develop embryoid-tike bodies.
  • Embryotd- like bodies ate noncontiguous dumps of polls that can grow on top of ail adherent layer of proliferating isolated placental cells»
  • the term “embryoid-like” is used because the clumps of cells resemble embryoid bodies, clumps of cells that grow from cultures of embryonic stem cells
  • Growth medium in which embtyoid-ltke bodies can develop ⁇ n a proliferating culture of isolated placentalcells includes medium comprising, c.g., DMEM-LG (e.g., from Gibco); 2% fetal calf eenira (e.g...
  • ITS tx raHilm-tramfemn-sckmum
  • LA-8SA lx linoleic acid-bovine serani albumin
  • 10 -9 M dexamethasone e.g.;from Sigma
  • 10 -4 M ascorbic acid 2- phosphate ie.g., from Sigma
  • epidermal growth factor 10 ng/mL
  • PDGF-BB platelet-derived growth fector
  • the isolated placental Cells, isolated raultipotehtplaeental cells or isolated placental stem cells, and populations of such isolated placental cells, useful in the methods disclosed herein, e.g., the methods of treatment of a DPN of a subject, are tissue culture plasiic- adherent human placental cells that have characteristics of mulnpotent cells or stem cells, and express a plurality of markers that can be used to identify and/or isolate the cells, or populations of cells feat comprise the stem cells.
  • fee PDACs are angiogenic, in vitro or in vivo.
  • the isolated placental cells, and placental cell populations described herein include placental cells and placental cell-containing cell populations obtained directly from the placenta, or any part thereof (e.g,, chorion, placental cotyledons, or the l ike)- Isolated placental cell populations also include populations of (that is, two or mom) isolated placental cells in culture, and a population m a container, e.g., a bag.
  • Placental Cells e.g., placental multipotent cells and placental cells, useful in the methods and compositions described herein are described herein and, e.g., m US. latent Nos, 7,311,904; 7,311,905 and 7,468,276: and in US; Patent Application Publication No. 2007/0275362, the disclosures of which are hereby incorporated by reference in their entireties.
  • the isolated placental cells are isolated placental stem cells, In certain Other embodiments, the isolated placental cells are isolated placental multipotent cells. In one embodiment, the isolated placental cells, e g, PDACs, are CD34', GD 10* and CD 105' as detected by tiow cytometry. In another specific embodiment, the isolated CD34 ;
  • placental cells have the potential to differentiate crizo cells of a neural phenotype, ceils of an osteogenic phenotype, and/or cells of a chondrogenic phenotype.
  • the isolated CD34' CD10 + CD105 ; placental cells ate additionally CD200’.
  • the isolated CD34 , CD 10% CD 105 ’ placental cells ate additionally CD45 or CD90 + .
  • die isolated CD34% CD10% CD 105 * placental cells are additionally CD45" and CD90' as detected by Sow cytometry-
  • the isolated CD34 , CD 10+ CD 105", CD20T placental cells are additionally CD90* or CD45% as detected by flow cytometry.
  • the isolated CD34' CD 10 % CD105' , CD200 + placental cells are additionally CD90* and CD45 ; as detected by flow cytometry, i.e., the cells ate CD34' CD10' CD45% CD90' CD105' and CD200'
  • said COM' , CD10% CD45 , CD90', CD105 ⁇ , CD200' cells am additionally CD80 and CD86 ,
  • said placental cells are CD34' CD10 , GD105' and CD200' and one or more of CD38' CD45 , GD80 , CD86 , GDI 33' HLA-DR,DP,DQ , S$BA3% SSEA4' CD29% CD44% CD73% CD90% CD 105", HLA-A,B,C% PUU% ABC-p% and/or OCT-4*, as detected by flow cytometiy, to other embodiments, any of the CD34 ,
  • CD 10* CD 105' cells described above are additionally one or more of CD29 VCD38 , CD44 ' , CD54' , SH3 + or SH4 ' - to another specific embodiment, the cells are additionally CD44 + , to anotherspeeiftc embodiment of any of the isolated CD34 , CD10*.
  • CD105 + placental cells above* the cells are additionally one or more of CD117, CD133", KDR (VEGFR2 ), HLA- A,B,C+ HLA-DP*DQ,DR Programmed Death- 1 Ligand (PDLl) + , or any combination thereof
  • the CD34 ⁇ , CD104* CDlOS* cel1 ⁇ 4 ate additionally one or more of CD13n CD29+, CD33 + CD38-, C D44+ CD45-, CD54+, CD62E-, CD62L-,
  • any pf the placental cells described herein are additionaHy ABC-p4, as detected by flow cytometry* or OCT-44 (POU5FB), as determined by RT-ECR, wherein ABC-p is a placenta-specific ABC transporter protein (also known as breast cancer resistance protein (BCRP) and as mitoxamrone resistance protein (MXR)), and QCT-4 1 ⁇ 2 toe Octaroer-4 protein (PO05P1),
  • any pf toe placental cells described herein are additionally SSEA3- or SSEA4-, as determined by flow cytometry, wherein SSEA3 is Stage Specific Embryonic Antigen 3, and S$EA4 is Stage Specific Embryonic Antigen 4.
  • any of the placental cells described harem are additionally SSEA3- and SSEA4-.
  • any of die placental cells described heroin are additionally one or more ofMHC-B (e.g.* HL A-A,B,C4), MHC-II- (e.g.HLA -DP,DQ,DR-) or HLA-G-.
  • MHC-l t e.g., BLA-A,B,C*
  • Preferred peculations of cells comprising the isolated placental celts, whereih the populations of celts comprise, e.g,, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 6 ⁇ 3 ⁇ 4 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% isolated GD104, GD105-4 and CD34- placental cells; that is, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95% or 98% of cells m saidpopuiation are isolated CDJOr, CD105* and CD34- placental cells.
  • the isolated CD34--, CD10+-, GplOS ⁇ placental cells areadditionally CD200+;
  • the isolated CD34 ⁇ ; CD 10*, CDI05*, CD20O* placental cells are additionally CD90+ or CD45- , as detected by flow cytometry,
  • CD 105*, CD20CH- placental cells are additionally CD90* and CD45-, as detected by flow cytometry.
  • any Of the isolated CD34-, GDI 0*, CD105+ placental cells described above are additionally one or more ofCD29*, CD38-, CD44*, CD54+, $H3+ or SH4 k In another specific embodiment, the isolated CD34-.
  • the isolated placental cells ate additionally one or mote Of CD13*, CD29*, CD334, CD38-, CD44* CD45-, CDS4*, CD62E-, CD62L-, CD62P-, SH3* (CD73*), SH4t (CD73*), CD80-, CD86-, CD90*, SH2* (CD105+), GDI 06/VCAM4, CD 117- GDI 44/VE- cadherinlow, CD184/CXCR4-, CD200H CD133-, OCT-4+, SSEA3TM, SSEA4TM, ABC*p+,
  • CD to*, GDI 05* cells are additionally CD13*, CD29+, CD33+, CD38-, CD44*, CD45- CD54/ICAM*, CD62E-, CD62L-, CD62P-, SH3* (CD73*), $H4*(CD73*), CD8O ⁇ vCD86 ⁇ , CD90*, SH2*(CD105*K CD106/VCAM*, CDU7 > CDi44/VE ⁇ cadheriolow ?
  • compositions described herein are Ohe of more. Or all. Of CD 1.04, CD294, C3 ⁇ 434-> CP38- , CD444 CD45-, GD54+, CD90+, SH24 SH34, SH4+, SSEA3-, SSEA4 ⁇ # OCT4+, and ABC- p3 ⁇ 4 wherein said isolated placental cells are obtained by physical and/of bnzymatic disruption of placental tissue.
  • the isolated placental cells are OCT-44 and ABC-p4 M another specific embodiment, the isolated placental cells am OCT-44 and CD34-, wherein said isolated placental cells have at least one of the Mowing characteristics: CD 104, CD29+* CD444, CD4S-, CD54t, CD904, $H34 $M44, $S£A3 ⁇ > and SSEA4-.
  • the isolated placental cells are OCT-44, CD34-, CD 104, CD29+, CD444, Cp45 ⁇ , CD544;, GD9CI4, SH3+, SH44, SSEA3-, add SSEA4-.
  • toe isolated placental cells are OCT-44, CD34-", SSEA3--, and SSEA4TM.
  • the isolated placental celts are OCT-44 and CD34-, and 1 ⁇ 2 either SH24 bf SH34.
  • the isolated placental tolls are OCT-44, CD34-, SH24, and SH34.
  • the isolated placental ceils are OCT-44, CD34-, SSEA3TM, and SSEA4-, and are either SH2+ or SH3-t .
  • the isolated placental cells are OCT-44 and CD34-, and either SH24 or SH34, and is at least one of CDfOt, CD294.
  • die isolated placental cells are OCT-44, CD34-, CD1CH-, CD294, CD44+ CD45 ⁇ , CD544, CD904,
  • the isolated placental ceils useful in the methods and compositions disclosed herein are S824, SH34, SH44 and OCT-44,
  • the isolated placental cells are CDiO*, CD20+* CD44+, CDS44 CD90+, CD34- CD45-, S ⁇ EA3 ⁇ , or SSEA4-:
  • the isolated placerital cells are SB24, SH34, SH44, SSEA3-T said SSEA4-.
  • the isolated placental cells are Sti24, SH34, SH4+, SSEA3- and SSEA4— , CD104, CD294, CD444- CD54+ CD904, OCT-44, CD34 TM orCD45 ⁇ .
  • the isolated placental cells useful in the methods and compositions disclosed herein areCDlO*, CD294 CD34-, CP444 > CD45-, CD544 CD904 7H2-L SH3*, and SH4+; wherein said isolated placental cells are additionally one or more Of GCT-4+/SSEA3- or SSEA4-.
  • isolatedplacental cells usefiil to the methods and
  • compositions disclosed herein are CD200* or HLA*G ⁇ .
  • the isolated placental cells ate CQ200+ add HLA-CK
  • the isolated placental cells are additionally CD73* and CD1054,
  • the isolated placental pells are additionally CD34-, CD38- or CD45-.
  • the isolated placental cells are additionally CD34-> C 038 ⁇ aod C D45 ⁇ .
  • said stem cells are CD34-, CD38-, CD45-, CD73+ and CD1O5+.
  • said isolated CD20O+ or liLA-G- placental cells facilitate the formation of embryoid4ike bodies in a population of placental cells comprising die isolated placental cells, under conditions that allow the formation of embryoid-like bodies.
  • the isolated placental cells are isolated away from placental ceils that are not stem orinitltipotent cells, in another specific embodiment, said isolated placental cells are isolated away from placental cells that do not display these markers.
  • a cell population useful in die methods and compositions described jbe*3 ⁇ 4m i$ a population of cells comprising, e.g., that is enriched for, CD200+, HLA-G- stem celts.
  • said population is a population of placental cells; In various embodiments, at least about 10%, at least about 20%, at least abont 30%, at least about 40%, at leas* about 50%, or at least about 60% of cells m said cell population are isolated CD200 K HLA-G- placental cells. Preferably, at least about 70% of cells in said cel l population are isolated CD200+, HLA-G- placental cells. Mine preferably, at least about 90%, 95%, or 99% of said cells are isolated CD200+* HLA-G- placental Cells, to a specific embodiment of the cell populations, said isolated CD20O+, HLA-G- placental cells are also CD73+ and CDI05+.
  • said isolated CD200+, HLA-G- placental cells are also GD34-, CD3S- or CD45-, to another specific embodiment, said isolated CD200*, HLA-G- placental cells a* also CD34-, CD38-, CD45-. CD73+ and CD 105+.
  • said cell population produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryouMfire bodies.
  • said cell population 1 ⁇ 4 isolated away from placental cells that are not stem cells, to another specific embocfimeni.
  • said Isolated CDiOOk HLA-G- placental cells are isolated siway from placental cells that db not display these markers.
  • the isolated placental cells tisefid to tire methods and
  • compositions described: hereto are CD73k CD1 OSk and CD200k to another specific embodiment, toe isolated placental cells ate HLA-0-. toanother specific embodiment, toe isolated placental cells are CDM ⁇ CD38- or CD4$ ⁇ > to another specific embodiment, toe isolated placental cells ate CD34-, CD3fi-abd CD45- toanother specific embodiment, toe isolated placenta) cells are CD34-, €D3$- « CD45-. and HIA-G ⁇ .
  • CD105k and CD2004 placental cells facilitate toe formation of one or more embryoid-like bothes in a population of placental cells comprising toe isolated placental cells, when toe population is cultured under conditions that allow the formation of embryoid-like bodies
  • the isolated placental cells are isolated axyay from placental cells that are not toe isolated placental cells, to another specific embodiment, the isolated placental cells are isolated away from placental Cells that do not display these markers;
  • a cell population useful to toe methods and compositions described herein i$ a popuWon of cdls comprising, e.g., that is enriched for, isolated CD73+, CD105k CD200+ placental cells.
  • at least about 10%, at least about 20%, at least about 30%; at least about 40%, at least about 50%, or at least about 60% of cells to said cell population are isolated CD73t, CD 105k CD20Pt placental cetis.
  • at least about 70% Of said cells in said population of cells are isolated CD734,
  • CD 105k 00200* placental cells to another embodiment, at least about 90%, 95% or 99% of cells in said population of cells are isolated CD73*, CD 105k CD200* placental cells, to a specific embodiment of said populations, die isolated placental cells are HtA-Ck toanother specific embodiment, toe isolated placental cells me additionally CD34-, CD38- or CD45-. to another specific embodiment, the isolated placental cells are additionally CD34-, CD38- and CD45-. to another specific embodiment, toe isolated placentid cells are additionally CD34 ⁇ y CD38k, CD45-', and HLA-G-..
  • said population of cells produces one or more embryoid-like bodies when cultured under conditions that allow toe formationof embryoid-like bodies, to another specific embodiment, said population of placental cells is isolated away from placental cells that are not stem cells, to another specific embodiment , said population of placental DCla is isolated away fitow placental cells that do not display these characteristics.
  • the isolated placental cells are one or mote of
  • the isolated placental cells are CDlti-f, CD29+, CD34-, CD38-, CD444, C P 45 ⁇ , CD544, CD904, SH24;, SH34, SH4t, SSE A3-, SSEA4-, and OCT-4*.
  • the isolated placental cells are CDl ⁇ t CD294, C?D34-, CD38-.
  • the isolated placental eelfe are CD10*, CD294, CP34r-, CD3# ⁇ , CD4> ⁇ , CD544, $B24 , Sip*, SH44 and QCT-44.
  • die isolated placental cells are CDI04, CD29+, CD34TM, CD38-, CD44+, CD45- CD544-, CD904, HLA-G-, SH24, SH3t, SH44.
  • the isolated placental cells are OGT-4-f and ABOp-h
  • the isolated placental cells are SH24pagate SB34, SH44 and OCT44.
  • the isolated placental cells are OCT-44% CD34-, SSKA3 -, and SSEA4-.
  • said isolated OCT-44 ⁇ , CD34-, SSEA3-, and SSEA4 " placental cells are additionally CTU04, CD29+, CD34-, CD444, CD45-, CD544, CD90+ SH24 , SH34, and SH4*.
  • the isolated placental cells ate OCT- 44 ⁇ andCD34-, and either SH34 or SH44-.
  • the isolated placental cells ate €034- and either CD1 tit , CD294, CD444 , CD544, CD90* or OCT-44.
  • the isolated placental ceils useful in the methods and compositions described berm are CD2QCH- and OCT-44 , to a specific embodiment, the isolated placenta! Cells are CD734 and €01054; to another specific embodiment, said isolated placental cells are HLA-G-. to another specific embodiment, said isolated GD200* 0CT44 placental cells are CD34 ⁇ ⁇ , CD38 « or CD45-. to another specific embodiment, said isolated 002004-, QCT4+ placental cells ate CD34-, CD38- and CD45-.
  • said isolated CD200* OCT44 placental cells are CD34-, CD38-, CD45- CD73*, CD 105* and HLA * G ⁇ .
  • the isolated CD200+, 0CT4-4 placental cells ffccilflate die production of one or more embryoid-like bodies by a population of placental cells that comptises the isolated cells, when the population is cultured muter conditions that allow the formation of embryoid-Uke bodies, to another Specific embodiment, said isolated CD200*» OCT44 placental cells are isolated away from placental cells that are not stem cells, to another spedfic embodiment, said isolated CD2004, OCT-4+ placental cells are isolated away trom placental cells that do not display these characteristics.
  • a cell population useful in toe methods and compositions described hetoto is a population of cells comprising, e.g staggering that is entiched for, 032004, QCT-44 placenta! cells, to various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells, in said cell population are isolated GD200*, OCT-44 placental cells, to another embodiment, at least about 70% of said celts am said isolated CD2QG4, GCT-4+ placental cells;, to another embodiment, at least about 80%, 90%, 95%, or 99% of cells to said cell population are said isolated CD2O0% OCT-44 placentai cells.
  • said isolated CD2004> OCT-44 placental cells are additionally CD734 and CD 1054-.
  • said isolated CD2904, 0CT-44 placentai cells are additionally HLA-G-.
  • said isolated CD2004, OCT-44 placental cells are additionally CD34-, CD38- mid CD45-.
  • said isolated CD2004, QCP44 placental cells are additionally CD3A-, CD38- , CD45-, CD734, CD 1054 mid HLA-G-.
  • the cell population produces one or more embryoid-like bodies when culttoed under conditions that allow toe formation of erohryoid-tike bodies.
  • said cell population is isolated away from placental cells that are not isolated CD2004 ; dCT-44 placental cells, to another specific embodiment, said cell population is isolated away from placental cells that do apt display these markers.
  • the isolated placenta! cells usefiil to the methods and compositions described herein are CD734, CD1054 and HLA-G ” .
  • the isolated CD73t, CDI054 and HLA-G- placental cells are additionally CD34-, GD38-- or CD45 to anodier specific embodiment, the isolated CD?3+v GD 1054-, HLA-G- placental cells are additionally CD04-, CJD38- and CD45-.
  • the isolated CD73+* CDi05+, HLA-G-placental cells are additionally OCT-44
  • the isolated C)E>734, CDlOS+v j3 ⁇ 4LA-(3 ⁇ placental cells are additionally CD2004.
  • the isolated CD734, CD105+, MLA-G- placental cells are additionally CD34-, CD3S- , CTM5 ⁇ ,OCT*44 and CD2004.
  • HLA-G- placental cells facilitate the formation of embryoid-like bodies in a population of placental cells comprising said cells, when die population is cultured under c ⁇ todition$ that aittiw the formation of embryoid-like bodies.
  • said the isolated CD73+, CD105+, HLA-G*- placental cells are isolated away from placental celts that are not the isolated CD73+, CD 1054 , HI A-GTM placental cells.
  • said the isolated CD73+, CD105+, HLA-G- ptacental cells are isolated away from placental cells that dp not display these markers.
  • * ceUpoptoation usefiit id is a population of cells comprising, e.g>, that is enriched for, isolated CD73+, CDlOSt and HLA*G ⁇ placental cells.
  • said isolated CD73+, €D105+, HLA-G- placental cells are additionally CD34-, CD3&- or CD45-.
  • said isolated CD73+, CDI05+, HLA-G- placental cells are additionally CD34-, CD3&- and CD45-, to another specific embodiment, said isolated CD73+, CD 105% HLA-GTM placental cells are additionally OCT-4+.
  • said isolated CD73+, CD 1G5+, HLA-GTM placental celts are additionally €D2O0% in another specific embodiment, said isolated CD73+, CD105% HLA-G- placental cells are additionally CD34-, CD3&-, CD45-, OCT-4+ and CD200+.
  • said cell population is isolated away from placental cells dim are not CD73+, CD1G5+, HLA-G- placental cells, to another specific embodiment, said cell population « isolated away from placental cells that do not display these markers.
  • the isolated placental cells useful in the methods and compositions described herein are CD73+ and CD1G5+ and facilitate the formation of one or mote embryoid-like bodies in a population of isolated placental cells comprising said CD73+,
  • CD IG5+ cells when said population 3 ⁇ 4 cultured tinder condition that allow formation of embryoid-like bodies.
  • said isolated CD73+, CD 105+ placental cells are additionally CD34-, C3D38-- or CD45- .
  • said isolated CD73+, CD105+ placental cells am additionally CD34-, CD38- and CD45-.
  • said isolated CD73+, CDi 05+ placental cells are additionally OCT-4+.
  • CD 105+ placental pells ate additionally ⁇ +, CD34-, CD3&- and CD45TM
  • said isolated CD73+, CD105+ placental cells ate isolated away from placental cells that are not said cells.
  • Said isolated CD73*, CD 105+ placental cells are isolated away from placental cells that do not display these characteristics.
  • a cell poptilation useful iii the methods and compositions described herein is a population of cells comprising, e.g > , that is enriched for # isolated placental cells that are CD73+; CD105+ and facilitate the formation of one or more embryokMike bodies in a population of isolated placental celts comprising said ceils when said population is cultured under conditions that allow formation of embryoid-like bodies» 10 various embodiments, at least about lb% # at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 00% of cells in Said population of cells ait said isolated CD73fj CD105+ placental cells.
  • # at least about 70% of cells in said population of cells are said isolated CD ' 73-f, CD 105+ placental Cells. In another embodiment, at least about 90%, 95% or 99% of cells in said population of cells are said isolated CD73+, CDIG5+ placental cells.
  • Said isolated CD73+, CD105+ p1 ⁇ 2cental cells are additionally CD34-, 0>3&- or CD45 v tn another specific embodiment, said isolated CD73+, CD 105+ placental cells are additionally CD34TM, CD3BTM and CD4S-.
  • said Isolated CT>73+ > CD105+ placental cells ate additionally OCT-44 ⁇ , In another specific embodiment, said isolated CD73+, CD105+ placental cells are additionally CD200+. In another specific embodiment, said isolated CD73+, CD105+ placental cells are additionally CD34-, CD38K CD45-", OCT-4+ and CD200+. In another specific embodiment, said cell population is isolated away from placental cells that are not said isolated CD73+, CD 105+ placental cells, in another specific embodiment, said cell populatitm is isolated away from placental cells that do not display these markers.
  • the isolated placental cells useful in the methods and compositions described herein are OCT-4+ and facilitate formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said ce lls when cultured under conditions that allow formation of embryoid-like bodies.
  • said isolated QCT-4+ placental cells are additionally CD73+ and CD105+.
  • said isolated QCT-4+placental cells are additionally CD34- , CD38-, Or CD4S ⁇ -
  • said isolated 0CT4* placental cells are additionally CD20O*.
  • said isolatedOCT-4* placenta! cells are additionally CD73*, CD!
  • said isolated OCT-4-5 placental celts are isolated away from placental celts that are not OCT-4* placental cells-
  • said isolated CfcT-4* placental cells are isolated away fiord placentaleells thai do not display these characteristics.
  • a cell population useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, isolated placental cells that are OCT-4* and facilitate the formation of one or nwe embiyoi ⁇ lijke bodies to a population of isolated placental cells comprising said cells when said population 1 ⁇ 4 cultured under conditions that allow formation of embryoid-lifce bodies, to various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least abdot 50%, or at least about 60% of cells iu said population of cells are said isolated OCT-44 placental cells.
  • At leasf about 70% of cells in said population of cells are said isolated OCT-4* placental cells. In another embodiment, at least about 80%, 90%, 95% or 99% of cells in said population of cells are said isolated OCT-4* placental celts.
  • said isolated OCT-4* placental cells are additionally CD34-, CD38- or CD45-.
  • said Isolated OCT-4* placental cells are additionally CD34-, CD38- and CD45-i
  • said isolated OCT-4* placental cells are additionally CD73+ and CD105*. to another specific embodiment, said isolated OCT- 4* placental cells ace additionally CD200*.
  • said isolated OCT- 4* placental cells are additionally CD73*, CD 105*, GD200*, CD34-, CD38 ⁇ , and CD45- to another specific embodiment, said cell population is isolated away ftoro plaeental celts that are not Said cells, to another specific embodiment; said cell population 1 ⁇ 2 isolated away from placental cells that do not display these markers.
  • the isolated placental cells usefiti 1 ⁇ 2 the methods and compositions described herein are isolated HLA*A,B,C* CD45 ⁇ vCD133 ⁇ and CD34 ⁇ placental cells
  • a cell population useful in the methods aud eompositions described herein is a population of cells comprising isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% of at least about 99% of cells in said isolatedpopnlation of cells are isolated ELA-A,B,C+, GD45 , CD 133- and GD34- placental cells.
  • said isolated placental cell or population of isolated placental cells to isolated away from placentaicells that are not HLA-A,B,0, CD45-, CD 133- and CD34- placental cells.
  • said isolated placental cells are non* maternal m origin .
  • said isolated population of placental cells are substantially free of maternal components; e.g,, at least about 40%, 45%, 5-0%, 55%, 60%*
  • the isolated placental cell! useful in the methods and compositions describedherein are isolated CDIOf, ⁇ 013- ⁇ €033+, CD45-, CDi l7- and CD 133- plaeenral cefls.
  • acell population useful in the methods and compositions described hereto is a population of ceils comprising isolated placental cells. whereto ar least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells to said population of celts are isolated CDI Or, CD! 3% CD334, CD45-,
  • CDl 17- and CDI 33- placental cells to a specific embodiment, said isolated placental cells or population of isolated placental cells to Isolated away from placental cells that are not said isolated placental cells, to another specific emboditoent, said isolated CDltH ⁇ , CD13+, CD33+, C045-, CDl 17- and CD133- placental cells ate non-matemal in origin, i.e., have the fetal genotype, In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells to said isolated population Of placental cells, are non-matemal in origin. In another specific embodiment, said isolated placental cells or population of isolated placental cells are isolated away from placental cells that do not display these characteristics,
  • the isolated placental cells useful to the methods and compositions described hereto are isolated CDl 0-, CD33- , CD44+, CD45- , and CD 117 - placental cells.
  • a cell population useful for the hi the methods and compositions described herein is a population of cells comprising, e.g., enriched for, isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells to said population of cells are isolated CD to*-, CD33-, CD44+, CD45 -, and CDl 17- placental cells, to a specific embodiment, said isolated placental cell or population of isolated placental cells is isolated away from placental cells that are not said cells.
  • said isolated placental cells am non-matemal in origin. In anoto specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, &%, 70%, ?5%, 90%, 85%, 90%, 95%, 98% or 99% Of Said cells in said cell population are non-matemal in origin. In another specific embodiment, said isolated placental cell or pqptdation of isolated placental cells is isolated away from placental cells that do not display these markers.
  • a cell population useful for in the methods and compositions described herein is a population of cells comprising, e.g,, enriched 3 ⁇ 4, isolated CD 10TM, CD ! 3-, CD33TM, CD45-, and CDU? ⁇ placental ce3 ⁇ 4 wherein at least about 70%, at least about 80%, at least abom 90%; at least about 95% or at least about 99% of cells in said population are CD 10-, CDI 3 ⁇ , CD33-, CD45TM, and CDI 17- placental cells.
  • said isolated placental dells Or population of isolated placental cells are isolated away from placental cells that are not said cells.
  • said isolated placental Cells are non-matemal in Origin.
  • at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are non-materaal in Origin.
  • said isolated placental cells or population of isolated placental cells is isolated away from placental cells 3 ⁇ 4t do not display these characteristics.
  • die isolated placental Cells useful to the methods and compositions described heroin are HLA ⁇ , ⁇ , ⁇ 3 ⁇ 4 €D45 ⁇ , CD34TM, and CD133TM, and ate additionally CD 10+, CD13+, CD38*, CD44t, CD9(Hv CD 1054, CD20CH and/or HLA-G-, and/or negative tor GDI 17.
  • a cell population useful in the methods described herein is a population of cells comprising isolated placental cells, wherein at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 98% car about 99% of fite cells in said population tune isolated placental cells that are HLA ⁇ , ⁇ , ⁇ , €D45 ⁇ , CD34 TM , CD133TM, and that are additionally positive for CD 10, CD13, CD38, CD44, CD90, CDI05, CD200, and/or negative for CDI 17 and/or HLA-G.
  • said isolated placental cells or population of isolated placental cells are isolated Sway 1 ⁇ 4ro placental cells that ate not said cells, !n another specific embodiment, said isolated placental cells ate non-matemal in origin, to another specific embodiment, at least about 40% ⁇ 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% Of said cells to said cel! population are non-matemal in origin.
  • said isolated placental cells or population of isolated placental cells are isolated away from placenta! cells that do not display these markets-
  • the isolated placental cells tisefid in the methods and compositions described herein are isolated placental cells that are CD2QQ4 and CD104, as determined by antibody binding, and GDI 17-, as determined by both antibody binding and RT- PCR.
  • the isolated placental cells useful in the methods and compositions described herein are isolated placental ceils, e,&, placental stent cells or placental muhipotentcells, that are CDKH-, CD29-, GD544, CD2004-, HLA-G-, MHC class 1+ and jh-2- microglobylin ⁇
  • cells usefulin the methods and compositions described herein are placental cells wherein the expression of at least one cellular marker is at least two-fbld higher titan tbra mesenchymal stem celt (e.g., a bone marrow-derived mesenchymal stem cell).
  • said isolated placental cells are non ⁇ maternal in origin.
  • at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are ⁇ -maiemal inorigin.
  • the isotered placental cells useful in the methods add compositions described herein are tsolated placental cells, e.g., placental stem cells or placental omnipotent cells, that ate tine or mote ofCDI0% CD29+, CD444, CD45-, CD54/1CAM4, CD62E-, CD62L-, CD62P-, CD8 ⁇ K CD86-, CD103-, CD104-, GDI 054, CD 1CWVCAM+ CD144/YE-cadherinjow, CD184/CXCR4-, p2 ⁇ nicroglobolintow, MHC-Ilow, MHC41-, HLA- Glow, and/or PDLllow.
  • placental stem cells or placental omnipotent cells that ate tine or mote ofCDI0% CD29+, CD444, CD45-, CD54/1CAM4, CD62E-, CD62L-, CD62P-, CD8 ⁇ K CD86-, CD103-, CD104-, GDI
  • the isolated placental ceils ate at least CD29+ and CD54K in another specific embodiment, the isolated placental cells ate at least CD44+ and CDI064, In another specific embodiment, the isolated placenta ⁇ cells ate at least CD29-K
  • a cell population useful in the methods and compositions described herein comprises isolated placental cells, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said cell population are isolated placental cells that are one or rooreof CD!On CD29+, CD44-f, GD45- , CD54/ICAM-r, CD62-E-, CD62-L-, CD62-P -,
  • at least 50%, 60%, 70%, 80%, 90%, >5%, 98% or 99% of cells to said cell population are CD10% CP29*, CD44+, CD45-r, CD54/ICAM% CD62-E- V CD62-L-, CD62-PTM, CD8G- CD$6- ⁇ , CDl 03- CD104- CD10S ⁇ , CD 106/VCAM ⁇ CDH4/VE- cadherindim, CD184/CXCR4 , p2-microglobulindim.
  • 3 ⁇ 4 ⁇ compositions described hereto are isolated placental cells tout are one or mote, or all, of CD10+-, CD29+, CD34-, CD38-, CD444, CD45-, CD544, CD904, 5 ⁇ 2*,$ ⁇ 3+, S1I4+, SSBA3-.
  • ABC-p to a placenta-specific ABC traitoporter protein also known as breast cancer resistance protein (BCRP) and as mtioxantrone resistance proteto (MXR)
  • BCRP breast cancer resistance protein
  • MXR mtioxantrone resistance proteto
  • die isolated placental cells useful in the methods of treatment provided hereto, can be distinguished from mesenchymal stem cells on toe bask of toe expression of one or more genes, ihe expression of which is significantly Mgher (that is, at least twofold higher) in the isolated placental cells than in an equivalent number of b ⁇ w matrow-derived mesenchymal stem cells, wherein toe one or more genes are ACTG2, ADARB 1, AMIG02, ARTS- 1, B4GALT6, BGHE, Cl lor&, CD20O, COMAL COL4A2, CPAADMD, DSC3, D$G2 r ELOVL2, 3F2RL1, FU10781, GATA6, GPR126, GPRC5B, HLA-G, 1C AMI, 1ER3, IGFBP7 V
  • ILIA ILIA, 1L6, it 18, KRT18, KRT8, LIPG, LRAP, MATNZ, MEST, NFE2L3, NUAKl, PCDH7, PDLIM3, PKP2, RTNl , SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21,TGFB2, VTRZC3M1 2A,m 8 wmbmation of any of the foregoing, when the celts are grown under equivalent conditions. See, e.g., U.S. Patent Application Publication Hb. 2007/0275362, the disclosure of which is incorporated heron by reference in 1 ⁇ 2 entirety.
  • said expression of said one ore more genes is detennined, e,g Berry by RT-PCR or mtcroainray analysis, using a VI 33-A tnicroarr&y (Asymetrix).
  • Said isolated placental celts express said one or mote genes when cultured for a number of population doublings, e.g., anywhere from about 3 to about 35 population doublings, in a medium comprising DMEM-LG (0.6-, from Gibco); 2% fetal calf serum (e.g,, from Hyclone Labs.); Ix insufin-transfemn-selenium (ITS); ix linoleic acid-bovineserom albumin (LA-BSA); 10-9 M dexamethasone (e.g , from Sigma); 104 M ascorbic acid 2-phosphafo (e.g , from Sigma); epidermal growth factor 10 ng/mL (DMEM-LG (0.6-,
  • NM_001615 (ACTG2X BC065545 (ADARBl), (NMJ 81847 (AM1G02), AY358590 (ARTS- 1), BC0T4884 (B4GALT6), BC008396 (BCHE), BC020196 (Cllorf9), BC03I 103 (CD20OX NM consider 001845 (CQL4AI ), MMJ301846 (COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (055C3), NMJ>01943 (DSG2X AF338241 (ELOVtJ), AY336105 (F2RLI), KMJH8215 (FUI0781X AY416799 (GATA6), BC075798 (GPR126), NMJU6235 (GPRC5B), AF340038 (1C AM I), BC000844 (1ER3), BC066339 (1GFBP7), 8CQ13142 (ILIA), BTO 19749 (IL
  • said isolated placental cells express each of
  • ACTG2* ADARBl, AM 1602, ARTS-1.
  • the placental cells express CD200 and ARTS! (amhopeptidase regulator of type i tumor necrosis factor); ARTS*!
  • LRAP leukocyte- derived arginine aminopeptidase
  • IL6 miterleukinrti
  • TGFR2 transforming growth factor, beta 2
  • ILti and KRTl 8 farnesoih 18
  • 1ER3 immediate early response 3
  • WEST mesoderm specific transcript homofog
  • CD20O and lER3 CD200 and 1L6; 00200 and KRT18; CD200 and LRAP
  • CD200 and MEST CD200 and NEI52L3 (nuclear factor (erythroid- derived 2>Hke 3); or CD20O and TGFB2 at a detectably higher level than an equivalent number of bone marrow-deri ved mesenchymal stem cells (BM-MSCs) wherein said bone marrow- derived mesenchymal stem cells have undergone a number of passages in culture equivalent to the number Of passages said isolated placental cells have undergone.
  • BM-MSCs bone marrow-deri
  • the placental cells express ARTS-1 , CD290, 1L6 and LRAP; ARTS-1 , IL6, TGFB2, IER3, KRT18 and MEST; €D20d, 1ER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; ARTS-l, CD200 t lER3, IL6.
  • probes based on the sequence of the gen ⁇ $ can be individually selected and constructed by conventional techni ques.
  • Expression of the genes can be assessed, e g., on a microarray comprising probes to one or more of the genes, e g., an Asymetrix GENECR1P® Human Genome U133A 2.0 array, or an Affymetrk GEMECTlIPS Human Genome U 133 Plus 2.0 (Santa Clara, California). Expression of these genes can be assessed even if the sequence for a particular GenBank accession number is amended because probes specific for die amended sequence can readily be generated using well-known standard techniques.
  • the level of expression of these genes can be used to confirm the identity of a population of isolated placental cells, to identify a population of cells as comprising at least a plurality of isolated placental cells, or foe like.
  • Populations of isolated placental celU, the identity Of which is confirmed can be clonil, e.g., populations of isolated placental cells expanded from a single isolated platseidal cell, car a mixed population of stem cells, e,gchev a population of cells comprising solely isolated placental cells that are expanded from multiple Isolated placental cells, or a population of cells comprising isolated placental cells, as described herein, and at least one other type of cell.
  • the level of expression of these genes can be used to select peculations of isolated placental cells, For example, a population of cells, e,g.. clonaHy-expandedcelis, may be selected if the expression of Ope or more of the genes listed above is significantly higher in a sample from the population tif cells than in an equivalent peculation ofmesenbhymal stem cells.
  • Such selecting cap be of a population from a plurality of isolated placen tal cell populations, fix»» a plurality Of cell populations, the identity of which is not known, etc.
  • Isolated placental cells can be selected cm foe basis of the level of expression of one ot mote such genes as compared tb the level of expression in said one or more genes in, e.g., a mesenchymal stem cell control, for example, the level of expression in said one or more genes in an equivalent number of bone marrow-derived mesenchymal stem cells.
  • IP one epibodipjent the level of expression of said one or more genes in a sample comprising an equivalent number of mesenchymal stem cells is used as a control.
  • the control for isolated placental cells tested under certain conditions, is a numeric value representing the level of expression of said one or more genes in mesenchymal stem cel ls under said conditions.
  • placental cells e.g., PDACs
  • said placental adherent cells are adherent to tissue culture plastic.
  • said population of celk induce endothelial cells to form sprouts or tube-like structures when cultured in the presence of an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) of basic fibroblast growth factor (bFGF), e g. , on a substrate such as MATRZGELTM
  • VEGF vascular endothelial growth factor
  • EGF epithelial growth factor
  • PDGF platelet derived growth factor
  • bFGF basic fibroblast growth factor
  • die PDACs provided herein, a population of cells, e.g., a population of PDACs, or a population of cells wherein at least about 50%, 60%, 70%, 80%,
  • the PDACs express increased levels ofCD202b ⁇ lL-8 and/or VEGF under hypoxic conditions (e.g,, less than about 5% Q2)compared to hoimoxic conditions (e.g., about 20% or about 21% 02),
  • any of the PDACS Oi poptitotiohs bf ceilb comprising
  • the PDACs described herein cad cause the formation of sprouts or tuhe-like structures to a population of endothelial cells to contact with said placental derived adherent cells
  • the PDACs are ccKwhured with human endothelial eeHs, whkh forovsprouts or tubolike structures, or support the formation of endothelial cell sprouts, e.g., when cultured to the presence of extracellular matrix proteins such as collagen type I and IV, and/or angiogenic factors such as vascular endothelial growth factor (VEGF), epithelial growth fector (EOF), platelet derived growth factor (PDGf) or basic fibroblast growth factor (bFGF), e.g., in or on a substrate suchas placental collagen or MATRIGELTM for at least 4 days.
  • VEGF vascular endothelial growth factor
  • EEF epithelial growth fector
  • PDGf platelet
  • any of the populations of cells comprising: placental derived adherent cells, described hereto, secrete angiogenic factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), or toterleukm-8 (IV8) and thereby can induce human endothelial cells to form sprouts or tube-like structures when cultured to the presence of extracellular matrix proteins such as collagen type 1 and IV e.g., in or on a substrate such as placental collagen or MATRIGELTM j
  • any of the above populations of cells comprising placental derived adherent cells (PDACs) secretes angiogenic factors.
  • PDACs placental derived adherent cells
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • PDGF platelet derived growth factor
  • hFGF basic fibroblast growth factor
  • H>3 ⁇ 4 vascular endothelial growth factor
  • the population of cells comprising PDACs secretes one or more angiogenic factors and thereby induces human endothelial cells to migrate jn an to v1 ⁇ 4o wound healing assay
  • foe population of cells comprising placental derived adherent cells induces maturation, differentiation or proiiferatioft of human endotheiial celts, endothelial progenitors, myocyte or myoblasts.
  • fOOlOSj The isolated placental cells described hereto display the above characteristics (e g,, combinations of cell surface markers and/or gene expression profiles) in primary culture, or during proliferation in medium comprising, e g., DMEM-LG (Gibed), 2% fetal calf sentm (PCS) (Hyclone Laboratories), lx insulimnansferrm-sdenium (ITS), lx tenolenic-acidrbovme ⁇ semm- albumin (LA-BSA), 10-9 M dexamethasone (Sigma), 10-4M ascotbic acid 2-phosphate (Sigma), epidermal growth factor (EGFUOhg/nil (k&D Systems), platelet derived-growth factor (PDC5F- BB) iOrtg/m) (R&D Systems), and jOOU penicillm/lOOOU streptomycin.
  • DMEM-LG Gibco-fetal calf sentm
  • PCS 2%
  • the cells are human.
  • the cellular marker characteristics of gene expression characteristics ate human markers or human genes.
  • said cells or population have been expanded, W example, passaged at least, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or more, or proliferated fdr at least, about, or nb mote than, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31,
  • said cells or population are primary isolates, in another specific embodiment of the isolated placental cells, or populationsof celiscomprising isolated placental cells, that are disclosed herein, said isolated placental cells are fetal in origin (that is, have the fetid genotype).
  • said isolated placental cells do not differentiate during culturing in growth medium, iejon medium formulated to promote proliferation, e.g., during proliferation in growth medium.
  • said isolated placental cells do not require a feeder layer tit order fb proliferate, in another specific embodiment, said isolated placental celts do not differentiate in culture in the absence of a feeder layer, solely because of the lack of a feeder cell layer.
  • cells usefiil in the methods and compositions described herein are isolated placental cells, wherein a plutatiiy of said isolated placental cells are positive ibr aldehyde dehydrogenase (ALDH), as assessed by an aldehyde dehydrogenase activity assay
  • ALDH aldehyde dehydrogenase
  • assays are known in the art (see, eg.
  • said ALDH assay nses Aldefluor® (Aldagen, Inc., Ashland, Oregon) as a marker of aldehyde dehydrogenase activity, lit a specific embodiment, said plurality is between about 3% and about 25% of cells in said population of cells.
  • Aldefluor® Aldefluor® (Aldagen, Inc., Ashland, Oregon) as a marker of aldehyde dehydrogenase activity
  • multipotent isolated umbilical cord cells wherein a plurality of said isolated umbilical cord celts are positive for aldehyde dehydrogenase, as assessed byan aldehyde dehydrogenase activity assay dial uses Aldefluor® as an indicatotofaldehyde dehydrogenase activity.
  • said plurality is between about 3% andabout 25% Of cells in said population of cells.
  • said population of isolated placental cells or isolated umbilical cord cells shows at least three- fold, or ar least fi ve-fold, higher ALDH activity than a population of bone marrow-derived mesenchymal shun cells having about the same number of cells and cultured under the same conditions.
  • the placental cells in $&d populations of cells are substantially free of cells having a maternal genotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 30%, 35%, 00%, 95%, 98% or 99% of the placental cells in said population have a fetal genotype.
  • the populations of cells comprising said placental cells are substantially free of cells having a maternal genotype; e.g,, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells In said population have a fetal genotype.
  • the karyotype of the cells is normal lu another specific embodiment of any of the above placental cells or cell populations, the cells, or cells in the population of cells, are non- maternal in Origin.
  • Isolated placental cells, or populations of isolated placental cells, bearing doy of the above combuiatiohs Of markets, can be combined in any ratio. Any two or more of the above isolated placental cell populations can be combined to form an isolated placental cell population.
  • an population of isolated placental cells caft comprise a first population of isolated placental cells defined by one pf the marker combinations described above, and a second population of isolated placental cells defined by another of the marker combinations described above, wheteiti said Hist atidsecond populations are combined in a ratio of about 1 ;99, 2:98, 3 ⁇ 497, 4:96, 5:95, 10:90,20:80, 30:70, 40160, 50:50, 6 ⁇ 4 ⁇ ; 70:30 r 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about 99.1, l « tike fashion, any three, four, five or more of tlie a ⁇ ove-described isolated placental celts or isolated placental cells populations cap be combmed
  • Isolated placental cells useful in the methods and compositions described herein cab be obtained, e.g., by disruption of placental tissue, with or without etwymatic digestion (See Section 4.2.3) or perfusion (see Section 4.2.4).
  • populations Of isolated placental cells can be produced according to a method comprismg perfhsing a mammalian placesia that has been drained of cord blood and perfused to remove residual blood; perfusing said placenta with a perfusion solution; and collecting said perfusion solution, wherein said perfusion solution after perfbsio» comprises a population of placental cells that coinprises isolated placental Cells; and isolating a plurality of said isdlated placemal cells from said population of cells.
  • die perfusion solution is passed through both the umbilical vein and umbilical arteries and collected after it exudes from the placenta
  • die perSsion solution is passed through the Umbilical veiiiabd collected from the Umbilical arteries, or passed through die umbiHcaiurtetiesand collected from the umbilical vein.
  • the isolated placental cells, contained within a population of cells obtained from perfusion of a: placenta are at least 50%, 60%, 70%, 80%,
  • the isolated placental cells collected by perfusion comprise fetal and maternal cells.
  • the isolated placental cells collected by perfusion are at least 50%, 60%.70%, 89%, 90%, 95%, 99% or at least 99.5% fbtal cells.
  • composition cotaprismg a population of the isolated placental ce1 ⁇ 4 as described herein, collected by perfusion, wherein said composition comprises at least a portion of the perfusion Solution used to collect the Isolated placentalcells-
  • Isolated populations of the isolated placental cells described herein can be produced by digesting placental tissue With a tissue-disrupting enzyme to obtain a population of placental cells comprising the cells, and isolating, or substantially isolating, a plurality of the placental cells from the remainder Of said placental cells.
  • the Whole, Or any part of, the placenta can be digested to obtain the isolated placental ⁇ ells described herein.
  • said placental tissue can be a whole placenta, an ammotic membrane, chorion, a combination of antoion and chorion, or a combination of any of the foregoing.
  • the tissue ⁇ isrupting enzyme is trypsin or coliagenase.
  • the isolated placental cells, contained within a population of cells obtained from digesting a placenta are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% oral least 99:5% of said population of placental celts.
  • Theisotated populations of placentalceUs describedabove* and populations of isolated placental cells generally, can comprise about, at least, or no more than l x L0 ⁇ 3 x 10 ⁇ 5
  • isolated placental cells e.g., as part of a pharmaceutical composition comprising placental stem celts.
  • Populations of isolated placental cells useful in the methods of treatment described herein comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, Of 99% viable isolated placental cells, &g., as deterntined by, e.g., trypan blue exclusion,
  • the cell cotiebtien composition can comprise any physiologi ⁇ &lly-aeceptable solution suitable for the collection and/or culture of cells, e.g., the isolated placental cells described hereto, for example, a saitoe solution (e.g., phosphate-buffered saline, Kteb’s solution, modified Kreb’s solution, Eagle’s solution, 0,9% Na0. etc ), a culture medium (e.g., DMEM. H.DMEM, etc,), and the like,
  • a saitoe solution e.g., phosphate-buffered saline, Kteb’s solution, modified Kreb’s solution, Eagle’s solution, 0,9% Na0. etc
  • a culture medium e.g., DMEM. H.DMEM, etc,
  • the ceil collection composition can comprise One or more components that tend to preserve isolated placental cells, that is, prevent the isolated placental cells from dying, or delay the death of the isolated placental cells, reduce the number of isolated placental cells m a population Of cells that die, or the like, from the time of collection to the time of cuttaring.
  • Such components can be, e.g., an apoptosis inhibitor (e.g via a caspase inhibitor or JNK inhibitor); a vasodilator (e.g,, magnesium sulfate, an anttoypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropitt, corticotropin-ieleosing hormone, sodium nitropmsside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sul&te, aphosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 241H-lnddh3 «yl)-3*pentylami00-maleimide, pyrrolidine dithjocarbamate, or clonazepam); a TNF-a inhibitor', and/or an oxygen-carrying perfiuorocarbon (e.g congestion perfluoro
  • the cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metallcptotease, a Serine pmtease, a neutral protease, an RNase, m i DNase, or the like.
  • tissue-degrading enzymes include, but are not limited to, collagenases (e.g., collagenase l, II, III or IV, a collagenase from Clostridium histolytiawuetc. ⁇ ; dispose, thermoiysin, ejastase, trypsin, UBERASE, hyaiuronidase, arid the like.
  • the cell collection composition can comprise a bacteriocidally or bacteriostaticaUy effective amount ofan antibiotic
  • the antibiotic is a macrolide (e.g., tobmmycm), a cephalosporin (e.g., cephalexin, cephractine, cefriroxime, celprozil, cefaclor, cefixime dr cefadroxti), a clarithromycin, an erythromycin, a penicillin (e.g. t penicillin V) or a quiMone (eg.. Ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc.
  • a macrolide e.g., tobmmycm
  • a cephalosporin e.g., cephalexin, cephractine, cefriroxime, celprozil, cefaclor, cefixime dr cefadroxt
  • the antibiotic is acti ve against Gram(4) and/or Gram(-) bacteria, e g., Pseudomonas aeruginosa, Staphylococcus auteus, and the like.
  • the antibiotic is gentamycin, e.g., about O:O05% to about 0,01% fw/V) in culftue medium ⁇ 00123 j
  • the ceil collection composition can also comprise one or more of the following compounds: adenosine (about I mM to about 50 mM); D-giucose (about 20 niM to about 100 mM); magnesium ions (about I mM to about 50 mM); a macromolecule of molecular weight greater man 20,000 dalfons, in ode embodiment praseitt in an amount sufficient to maintain endothelial integrity and cellular viability (e.g-, a synthetic or naturally occurring colloid, a polysaccharide such as dextran
  • N-acetylcysteine present at about 0.1 mM to about 5 mM
  • an agent that prevents calcium entry into cells e.g., verapamil present at about ' 2 ⁇ to about 25 pM
  • nitroglycerin e.g., about 0.05 g/L to about 0.2 g/L
  • an anticoagulant in one embodiment, present in an amount sufficient to help prevent clotting of residual blood (e.g., heparin dr hirudin present at a concentration of about lOOO unitsd to about 100,000 units/!
  • an amiloride cpniauiing compound e.g., amiltiride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 ⁇ to about 5 ⁇ ).
  • a human placenta is recovered shortly after its expulsion after birth, in a pteferred embodiment, the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta. Preferably, Hie medical history continues after delivery.
  • a medical history can be used to coordinate subsequent use of me placenta or the isolated placental cells harvested therefrom.
  • isolated human placental cells can be used, in light Of the medical history , for personalized medicine for die infant associated with the placenta, or for parents, siblings or other relatives of the infant
  • the umbilical cord blood and phcental blood are preferably removed.
  • the cord blood m the placenta is recovered.
  • the placenta can be subjected to a conveotional cord blood recovery process, Typically a needle dr cannula is used, with the aid of gravity, to exsanguinate tire placenta (see, e.g., Anderson, U.S. PaienfNo. 5,372,581; Hessel et al., U,S, Patent No,
  • the needle or cannula 1 ⁇ 2 usually placed in the umbilical vein and the placenta can be gentlymassaged toaid in draining cord blcxKl from the placenta.
  • cord blood recovery may be performed commercially, e,g., LifeBank USA, Cedar Knolls, Hi.
  • the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
  • a placenta is transported frorothe delivery or birthing room to another location, eg,, a laboratory, for recovery of cord blood and collection of stem cells by, eg., perfusion or tissue dissociation.
  • the placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28 3 ⁇ 4 C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed 1 ⁇ 2 un insulated container.
  • the placenta is transported bin cord blood collection kit substantially as described in pending United States Patent No.7, 147,62b, die disclosure of Which is imrotporated by reference herein.
  • the placenta is delivered to foe laboratory four to twenty-four hours following delivery in certain embodiments, foe proximal umbilical cord is clamped, preferably within 4-5 cm (centimeter) of foe insertion into the placental disc prior to cord blood recovery, In other embodiments, foe proximal umbilical cord 1 ⁇ 4 clamped after cord blood recovery but prior to further processing of foe placenta,
  • the placenta prior to cell collection, can be stored trader sterile conditions trad at either room temperature or at a temperature of 5°C to 25 8 C.
  • the placenta may be stored for a period Of for a period of four to twenty-four hours, up to forty-tight horns, or longer than forty eight hours, prior to perfusing the placetita to remove any residual cord bibod.
  • foe placenta is harvested from between about zero hours to about two hours post- expulsion.
  • the placenta is preferably stored in an anticoagulant solution at a temperature of 5°C to 25*C. Suitable anticoagulant solutions are well known in foe art.
  • the anticoagulant solution comprises a solution of heparin (e.g,, 1% w/w in 1:1000 solution) .
  • the exsanguinated placenta is preferably stored for no more than 36 hours before placental cells are collected- f 00128)
  • the mammalian placenta or a part thereof, once collected and preparedgeneraily as above » can be treated in any art-known manner, can be perfused or disrupted.
  • ⁇ r g., digested with one or more tissue-fosrupting enzymes, to obtain isolated placental cells. 4.23 Physical Disruotioif and Enzymatic Digestion of H ⁇ ,.
  • stem cells are collected from a mammalian placenta by physical disruption of part of all Of the organ.
  • the placenta, or a portion thereof may be,e,g., reckon crusted, sheared, minced, diced, chopped, macerated or the like.
  • the tissue can then be cultured to obtain a population of isolated placental cells.
  • S disrupted using, e.g., culture medium, asaime solution, or a stem cell collection.
  • the placenta can be dissected into components prior to physical disruption and/or enzymatic digestion and stem cell recovery.
  • Isolated placental cells can be obtained from all or a portion qf ite amntotic membrane ⁇ chorion, umWcal cord, placental cotyledons, or any combination thereof, including from a whole placenta.
  • isolated placental cells are Obtained from placental tissue comprising amnion and chorion.
  • isolated placental cells can be obtained by disruption Of a small block of placental tissue, e.g., a block of placental tissue that is about 1, 2, 3, 4 » 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900 of about 1000 cubic millimeters in volume.
  • Any method of physical disruption can be used, provided that the method of disruption leaves a plurality, mote preferably a majority, and more preferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable, as determined by, e.g., trypan blue exclusion.
  • the isolated adherent placental cells can generally be collected from a placenta, or portion thereof, at any time within about the first three days post-expulsion, but preferably between about 8 hours and about 18 hours post-expulsion.
  • the disrupted tissue is cultured in tissue culture medium suitable for the proliferation of isolated placental cells.
  • isolated placental cells are collected by physical disruption of placental tissue, whereto the physical disruption includes enzymatic digestion, which can be accomplished by use of one or more tissue-digesting enzymes.
  • the placenta, or a portion thereof may also be physically disrupted and digested With rate or mote enzymes, and the resulting material then immersed to, or mixed into, a cell collection composition.
  • a preferred cell collection competition comprises one or more tmue-disniptive enzyme(s).
  • Enzymes that can be used to disrupt placenta tissue include papain, deoxyribonticleases ⁇ serme proteases, suchas trypsin, ohymbtrypsin, collagenase, dtspase or eiastase.
  • Smite proteases may be inhibited by alpha Z microglobulm in serum and therefore the medium used for digestion Is usually serum-foee.
  • BDTA and PNase are commonly used in enzyme digestion procedures to increase the efficiency of celt recovery.
  • the digestate is preferably diluted sous to avoid trapping cells within the viscous digest
  • Typical concentrations For digestion using trypsin include, 0:1% to about 2% trypsin, e.g,. about 0.25% trypsin.
  • Proteases can be used in combination, that is, two or more proteases in the same digestion reaction, or can be used sequentially in order to liberate placental cells, e.g., placental
  • a placenta, or part Seiedf. is digested first with ad appropriate amount of cotlagenase t at about 1 to about 2fng/tnl for, e.g,, 30 minutes, followed by digestion with trypsin, at a concentration of about 0.25%, for, e.g., 10 minutes, at 37*C.
  • Serine proteases are preferably wd consecutively following use of other enzymes.
  • the tissue cab further be disrupted by the addition of a chelator, e.g , ethylene glycol bis(2-ammoethyf efoer)-N,N,N ⁇ MMetraaceticacid (EOTA) or ethylenediaminetetraacetic acid (EDTA) to the stem cell collection composition comprising the stem cells, or to a solution ih Which the tissue is disrupted and/or digestetiprior to isolation df the stem cells with the stem cell collection composition.
  • a chelator e.g , ethylene glycol bis(2-ammoethyf efoer)-N,N,N ⁇ MMetraaceticacid (EOTA) or ethylenediaminetetraacetic acid (EDTA)
  • the digestate is washed, for example, three timeswifo culture medium, and the washed cells are seeded into culture flasks. The cells are then isolated by differential adherence, and characterized for, e.g., viability, cell surface markers, differentiation, and foe like.
  • the placental cells isolated can comprise a mix df placental cells derived from both fetal and maternal sources.
  • a portion of foe placenta that comprises Mb, or a negligible number of, maternal cells (for example, amnion) foe placental cells isolated therefrom will comprise almost exclusively fetal placental cells (that is, placental cells having foe genotype of the fetus).
  • Placental cells e.g., the placental calls described in Section 4.1, above, can be isolated from disrupted placental tissue by differential trypsihization ⁇ see Section 4.2.5, below) followed by culture in one or mote new ctilttite tontainers in fresh proliferation medium, optionally folJowedby a seccmddifferential ttypsinization step.
  • Placental cells can also be obtained by perfusion of the mammalian placenta.
  • Methods of perfusing mammalian placenta to obtain placenta! Cells are disclosed, e.g., iu Hariri, U.S. PaietuNos, 7,045,148 and 7,255,729, 1 ⁇ 2 Li.$.
  • Patent Application Publication Nos.2007/0275362 and 2007/0190042 the disclosures of each of which are incorporated herein by reference id their entireties;
  • Placental cells can be collected by perfUsion, e.g., through the placental vasculature, using, e.g., a cell collection composition as a perfusion solution.
  • a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein.
  • the flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta.
  • the perfusion solution is forcedtbrough the placenta using 8 pump, e.g.,a peristaltic pump.
  • the umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected b a sterile connection apparatus, such as sterile tubing. Hie sterile connection apparatus is connected to a perfusion manifold. j 00142)
  • tiie placenta is preferably oriented (e.g., suspended) in such a manner that the umbihcal artery and umbilical vein are located at the highest point of the placenta.
  • the placenta can be perfused by passage of a perfusion fluid through tie placental vasculature and surrounding tissue.
  • the placenta can also be perfused by passage of a perfhsion fluid into the umbilical vein and collection from tie umbilical arteries* or passage of a perfusion fluid into the umbilical arteries and collection from the umbilical vein.
  • the umbilical artery arid the umbilical vein are counected simuhaneqitoly, e.g., to a pipette that is connected via a flexible connector to a reservoir of tie perfusion solution»
  • the perfusion solution is passed into the umbilical vein and artery.
  • the perfusion solution exudes from and/or passes through the walls of tie blood vessels into tie surrounding tissues of the placenta, and is collected in a suitable open vessel from the surihee of foe placenta that was attached to foe uterus of the mother during gestation.
  • the perfusion solution may Slso be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in foe wall of the placenta which interfacedwith theroaternal Uterine wall.
  • Placental cells foat are collected by this method, which can be referred to as a “pan” method # ate typically a mixture of fetid and maternal cells #
  • the perfusion solution is passed through foe umbilical veins and collected from foe umbilical artery, or is passed through foe umbilical artery and collected from foe umbilical veins.
  • Placental cells collected by this method which CM be referred to as a “closed circuit” method, are typically almost exclusively fetal.
  • foe pan method that is. Whereby perfusate is collected after it has exuded from the maternal stde offoe p!acehta, results in a mix of fetal and maternal cells.
  • foe cells collected by this method can comprise a mixed population of placental cells, e.g#, placental stem cells or placental multipotem cells, of both fetal and maternal origin.
  • perfusion solely through foe placental vasculature In the closed circuit method, whereby perfusion fluid is passed through one or two placental vessels and is collected solely through foe remaining vessels), results in foe collection of a population of placental cells almost exclusively of fetal origin.
  • the closed circuit perfusion method can, id one embodiment, be performed as follows.
  • a post»partum placenta is obtained within about 48 hours after birth.
  • the umbilical cord is clamped and cut above foe clamp.
  • the umbilical cord cah be discarded or can processed to recover, e * g., «mbiUcai cord stem ce3 ⁇ 4 and/or tp process foe umbilical cord membrane for foe production of a biomaterial.
  • the amniotic membrane can be retained during perfosion, or can be separated from foe chorion, e.g., using blunt dissection with the fingers.
  • k can be, e g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce,, e.g,, an amniotic membrane biomaterial, e g,, foe bidmaterial described in U S. Application Publication No. 2004/0048796, the disclosure of which is ineorpofatedby reference herein in its entirety.
  • the umbilical cord vessels are exposed, e.g,, by partially cutting the umbilical cord membrane to expose a cross-section of the cord.
  • the vessels are Identified, and opened; eg., by advancing a closed alligator clamp thtoughthe cut end of each vessel
  • Theapparatus eg., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries.
  • the pump cait be any pttmp sttitable for the purpose, e g., a peristaltic pump.
  • Plastic tubing connected to a sterile collection reservoir, e g., a blood bag such as a 250 mL collection bag, is fee» inserted into fee placental vein, Alternatively, fee tubing connected to fee pump is inserted into fee placental vein, and «fees to a collection reservoir(s) are inserted ifeo one or both of the placental arteries.
  • the placenta is then perfused with a volume of perfusion solution, e.g ⁇ , about 750 ml ofpetfeston solution. Cells in the perfusate are then collected, e.g,, by centrifugation.
  • the placenta is periused with perfuSion sohmon, e.g., 100-300 mL perfesion solution, to remove residual blood prior to perfusion to collect placental cells, e.g., placental stem cells and/or placental muitipotent cells.
  • placenta is nbt perfused wife perfusion solution to remove residual blood prior to perfusion to col lect placental cells .
  • the proximal umbilical cord is damped during perfusion, and mom preferably, is damped Within 4-5 cm (centimeter) of fee cord's insertion into fee placental disc.
  • the first collection Of perfusion fluid from a mammalian placenta during the exsanguinattoh process is generally colored wife residual red blood cells of fee cord blood and/or placental blood.
  • the perfusion fluid becomes more colorless as perfiision proceeds and the residual cord blood cells- ate washed out oftheplacenta.
  • flrotn 30 to 100 ml (milliliter) of perfusion fluid is adequate to initially exsanguinate the placenta, but more or less perfusion fluid may be used depending On fee observed results,
  • the volume of perfusion liquid used to isolate placental cells may vary depending upon fee number of cells to be collected, fee size of the placenta, fee number of collections to be made from a single placenta, etc, h various embodiments, fee volume of perfusion liquid may be from 50 mL to SOOO mL, SO ntLto 4000 mL, 50 mL to 3OO0 ntL, 100 mL to 2000 mL, 250 mL to 2000 ml, SOO mL to 2000 mL, or 750 mL to 2000 mL.
  • the placenta is perfused wife 700-800 mL of perfusfeti liquid folldwmg exSatiguination.
  • the placenta can be perfused a plurality of times over fee course of several hours or several days. Where, the placenta is to be perfused a plurality of times, it may be maintained or cuitwred under aseptic conditions in a container or other suitable vessel, and perfused with the cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered salhe f ‘PBS”)) with or Without an anticoagulant (e g., heparin, warfarin sodium, coumarin, btshydroxycmimarin), and/or with or without ait antimicrobial agent (e.g., ⁇ * mercaptoethanol (0,1 mM); antibiotics such as streptomycin (e.g., at 40-100 pg/tul), penicillin
  • an isolated placenta is
  • the perfused placenta can be maituaihed for one or more additional timrfs), e,g., t, 2, 3, 4, 5, 6, 7, 8 » 9, 10, 11, 12, 13, 14, 15, 16, 17, i 8, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e,g token 700-800 tuL perfusion fluid.
  • the placenta can be perfused 1 , 2, 3, 4, 5 or more times, tor example, once every 1 , 2, 3 , 4, 5 or 6 hours.
  • perfusion of the placenta and collection of perfusion solution e.g., Cell collection composition
  • perfusion solution e.g., Cell collection composition
  • the perfusates at different time points can be further processed individually to recover time-dependent populations Of cells, e g,, Stem cells: Perfusates from dirfereni time points can also be pooled.
  • placental cells are collected at a time or times between about 8 hours and about I S hows posr-expulsiom
  • 1001511 Perfusion preferably results in the collection of significantly more placental cells than rite number obtainable from a mammalian placenta not perfused with stud solution, and not otherwise seated to obtain placental cells (e.g , fey tissue disruption, e.g. t enzymatic digestion).
  • ‘‘significantly more” means a» least 10% more.
  • Perfusion yields significantly mmre placental cells than, e.g.* the number of placental cells isolatable from culture mediant in which a placenta, or portion thereof has been cultured.
  • J0Q152J Placental cells can be isolated from placenta by perfusion with a solution comprising one car mote proteases pr other tissuenfistuptive enzymes.
  • a placenta or portion thereof e.g., amniotic membrane, amnion and chorion, placental lobule or cotyledon* Umbilical cord, or combination of any of the foregoing
  • is brought to 25-37T is brought to 25-37T, and is incubated with one or more tissne-disraptive enzymes in 200 mL ofa culture medium for 30 minutes.
  • Cells frdm toe perfusate are collected, brought to 4°C, and Washed with a cold inhibitor mix comprising 5 mM EDTA.2 mM difoiothreitol and 2 mM bete-merCaptoefoanol.
  • the placental cells are washed after several minutes with a cold (e ⁇ *, 4°C) stem cell collection composition.
  • the isolated placental cells the cells described *tt Section 4, 1 * above, whether obtained by perfusion or physiol disruption, e.g,, by enzymatic digestion, can initially be purified from (/.e.,be isolated from) other cells by Ficoll gradient centrifugation. Such centrifugation can follow any standard protocol for centrifugation speed, etc.
  • cells hoisted from the placenta areiecovered frofo perfttsate by ttentrifugatioh at 5000 it g for 15 minutes at room temperature, which separates cells from, e.g., c1 ⁇ 2tamtnating debris and platelets *
  • placental perfusate is concentrated to about 200 ml, gently layered over ficoll, and centrifuged at about 11 P0 x g for 20 minutes at 22% and the low-density interfile layer of cells is collected for further processing.
  • Cell pellets can be resuspended in fresh stem cell collection composition, or a medium suitable for cell maintenance ⁇ e.g., stem cell maintenance, for example, 1MDM serum- free medium containing 2U/ml heparin ahd 2 mM EDTA (GibcoBRL, NY). Die total mononuclear celt fraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo, Norway) according $0 the manufacturer's recommended procedure. ftiOlSS) Placental cells obtained by perfusion or digestion can.
  • stem cell maintenance for example, 1MDM serum- free medium containing 2U/ml heparin ahd 2 mM EDTA (GibcoBRL, NY). Die total mononuclear celt fraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo, Norway) according $0 the manufacturer's recommended procedure.
  • ftiOlSS Placental
  • the isolated placenta! celts which are tissue culture plastic-adherent, typically detach from the plastic surfaces within about five itimutes whereas other adherent populations typically requite more than 20-30 minutes incubation.
  • the detached placental Celts can be harvested following trypsinization and trypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS, Cambrexl ⁇ one embodiment of 1 ⁇ 2iation of adherent cells, aliquots of, for example, about 5- 10 x W cells are placed in each of several T-75 flasks, preferably fibronectin-coated T75 flasks.
  • foe cells can he cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Ourabrex), and placed in a tissue culture incubator (37% 5% CD2).
  • MSCGM Mesenchymal Stem Cell Growth Medium
  • non-adherent cells are remoVed frOm the flasks by waslihg with PBS.
  • the PBS is then replaced by MSCGM Flasks are preferably exaromeddaily for foe presence of various adherent cell types and m particular, for identification and expansion of clusters of fibroblastoid cells .
  • the number and type of cells collected from a mammalian placenta can he monitored * for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flcw cyfotnetiy, cell sorting, immimocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated ceil sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the ait, such as ICR and gene expression profiling. These techniques can be used, too, to identify cells that ate positive for one or mote particular markers.
  • FACS fluorescence activated cell sorting
  • MCS magnetic activated ceil sorting
  • one cap determine, using the techniques above, whether a Cell comprises a detectable amount Of CQ34; if so, the cell is CD34+: Likewise, if a cell produces enough OCT-d RNA to be detectable by RT-FCR, or significantly more OCT-4 RNA than an adult cell, foe cell 1 ⁇ 4 0CT-4+.
  • Antibodies to cell surface markers e,g. y CD markers snch as CD34
  • sequence ofstem cell-specific genes such as OCT-4
  • Placental cells particularly cells that have been isolated by Ficoll separation, differential adherence, or a combination of both, may be sorted using a fluorescence activated cell sorter (FACS), Fluorescence activated celt sorting (FACS) is a well-known method for separating particles, including cells* based an the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, ⁇ 51;150-1 ⁇ 45). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture.
  • FACS Fluorescence activated celt sorting
  • cell SiufacO marker-specific antibodies or ligands are labeled with distinct fluorescent labels.
  • Cells are processed through the cell sorter, allin1 ⁇ 2g separation of cells based on their ability to bind to foe antibodies used, FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plateS to facilitate separation and cloning.
  • cells from placenta e.g., PDACs are sorted on the basis of expression of one or moreof the markers CD34, CD38, Cb44, CD45, CTO, CPJOS, OCT-4 and/or HiA-G.
  • tissue culture plastic adherence selection can be accomplished before or after sorting: on toe basis of market expression, to one embodiment for example, cells are itorted first On the basis of their expression of CD34; CD34- cells are retained, and CD34- cells that are additionally CD20CM and HLA-G- are Separated from all other CD34 - cells, to another embodiment, cells ftom placenta ate sorted based bn their expression of markers CD2QO and/or HLA-G; for example, cells displaying CD20G and lacking BLAHS are isolated tor further use.
  • Cells that express, e.gchev CD200 and/or lack, e.g., HLA-G can. in a specific embodiment, be further sorted based on their expression of CD73 and/or CD 105, or epitopes recognized by antibodies SH2, SH3 or SH4, or lack of expression bfPD34; CD38 or GD45.
  • placental cells ate sorted by expression, or lack thereof, of CD200, HLA-& GD?3, CD! 05, CD34, GD38 and CD45, and placenta) cells that are CD200+, HLA-G-, CD73+.
  • CD105+, CD34-, CD38- and CD45- are isolated ftom otoer placental cells tot further use;
  • Placental cells can be sorted by one or mom of any of the markers described in Section 4, 1, above.
  • placental cells that are (1) adherent to tissue culture plastic, and (2) CJDtlOz, CD34- and CD 105f m sotted ftotti (i.e., isolated from) other placental cells
  • placental cells that are (1 ) adherent to tissue culture plastic, and (2) CD ' IO* ⁇ , CD34-, CD 105+ and CD200+ are spirted from (i.e., isolated from) other placental cells.
  • placental cells that are ( 1 ) adherent to tissue culture plastic; and ⁇ 2) CtN 0*, CD34-, CD45-, C»9 ⁇ H% CD165+ and CD2P04 ⁇ are sorted from (i.e., isolated from) other placental cells.
  • sequences for the markers listed herein are madily available in pubticiy-available databases such as GenBank or EMBL
  • any antibody, specific tor a particular marker can be used, in combination with any fluorophore or either label stiitabtefor the detection and sorting Of cellSje.g., fluorescence-activated cell sorting).
  • Autibody/titiorophore combinations to specific markers include, hut are not limited to, fluorescein isothiocyanate (FlTC) conjugated monoclonal antibodies agaihst HLA ⁇ i (availabk fiom Serotec, Raleigh, North Carolina), GDI 0 (available torn BD Immanocytometry Systems, San Jose, California), CD44 (available tom BD Biosciences Pbarmiogett, Sati Jose, California), and CDiOS (available ftorn R&D Systems Inc., Minneapolis, Minnesota); phycoerythrcn (PE) conjugated monoclonal antibodies against CD44, CD200, CDI 17, and CD 13 (BD Biosciences Phamtihgen); phycO «ythrin-Cy7 (PE Gy7) conjugated monoclonal antibodies against CD33 and CDi O (BD Biosciences Pharmingen); allophycocyanin (APC) conjugated streptavidm and monoclonal antibodies against CD38 (
  • antibodies that Can be used include, bttt are W limited to, CD133- AFC (Mihenyi), KDR-Siotin (CD309, Abeam), CytokeratinK-Fitc (Sigma or Dakti), HLA ABC-Fitc (BD), HLA DR,DQ,DP- PE (BD), ⁇ -2-microgIobulin-PE (BD)* CD80-PE (BD) and CD86-APC (BD).
  • CD4$-PerCP peridin chlorophyll protein
  • CD44-PE perdin chlorophyll protein
  • CD 19- PE CD tO-F (fluorescein); HLA-G-F and 7- amiuo-aetjnomycinfD (7-AADX HEA-ABOF; and the like ⁇
  • CD4$-PerCP peridin chlorophyll protein
  • CD44-PE perdin chlorophyll protein
  • CD 19- PE CD tO-F (fluorescein)
  • HLA-G-F 7- amiuo-aetjnomycinfD (7-AADX HEA-ABOF; and the like ⁇
  • This bat is not exhaustive, and other antibodies from Other suppliers are also commercially available.
  • the isolated placental celts provided herein can be assayed for CDI 17 or CD133
  • placental cells cati be labeled with an antibody to a Single marker add defected anti/sotted. Placental cells can also be simultaneously labeled with multiple antibodies to different markers.
  • magnetic beads can be used to stearate cells.
  • the cells may be sorted using a magnetic activated cell sorting (MACS) technique, a method for separating coarseles based on their ability to bind magnetic beads (05* lOd pm diameter).
  • MCS magnetic activated cell sorting
  • a variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizesa particular call surface molecule or hapten.
  • the beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker, 1 ⁇ 2 erne embodiment, these cells can then isolated and remtixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtker dishes for clonal isolation.
  • Isolated placental cells can also be charactenzed and/Or sorted based on cell morphology and growth characteristics.
  • isolated placental cells can be characterized as having, and/or selected on the basis of, e.g,, a fibroblastoid appearance in culture.
  • the isolated placental cells can also be characterized as having, ahd/or be selected, cm the basis of their ability to form embryoiddike bodies: in one embodiment, for example, placental cells that are fibroblastoid in shape, express CD73 and CD 105, and produce one or more embryoi ⁇ Mike bodies 1 ⁇ 2 culture are isolated from other placental cells.
  • OCT-44 placental cells that produce one or more embryotd-like bodies in culture are isolated from etoer placental cells.
  • isolated placental cells can be identified and characterized by a ctiloiiy forming unit assay.
  • Colony forming unit assays are commonly known in the art, such as MesenCultTM medium (Stem Cell Technologies, Inc., Vancouver British Columbia),
  • the isolated placental cells can be assessed for viability, proliferation potential* and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate uptake assay, pmpidium iodide uptake assay (to assess viability); and thymidine uptake assay, ⁇ (S ⁇ -tiimdhylthiazol-i-ylj-Z ⁇ -diphenyUetrazolium bromide) cell proliferation assay (to assess proliferation).
  • Longevity may be determined by methods well know# in toe art, such as by determining toe maximum dumber of population doubling in an extended culture.
  • Populations of isolated placental cells can be isolated directly from one or more placenta ⁇ that 1 ⁇ 4 the cell population can be a population of placental cells comprising the isolated placental cells, wherein the isolated placental cells are obtained from, or contained within, perfusate, or obtained horn, or contained wititith disrupted placental tissue, e.g ⁇ , placental tissue digestate (that; is, the collection of ceils obtained by enzymatic digestion Of a placenta or part thereof).
  • the isolated placental cells described herein can also be cultured and expanded to produce populatkmsof the isolated placental cells.
  • Populations of placental cells comprising the isolated placental cells can also be cultured and expanded to produce placental cell populations.
  • Placental Cell populations useful in the methods of treatment comprise the isolated placental cells, for example, the isolated placental cells as described in Section 4.1 herein, to various embodiments, at least 10%, 20%, 30%, 40%, $ ⁇ %, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a placental cell population are the isolated placental cells. That is, a population of the isolated placental cells can comprise, e,g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, We, 90% cells that are not the isolated placental cells.
  • Isolated placental cell populations useful in the methods ami compositions described herein cad be produced by, e.g., selecting isolated placental ceB, whether derived from enzymatic digestion or perfusion, drat express particular markers and/br particular culture or morphological characteristics.
  • a method of producing a cell population by selecting placental cells that (a) adhere tb a substrate, and (b) express CD200 and lack expression of HLA-G; and isolating said cells from other cells to form a cell population to another embodiment, a cell population « produced by selecting placental ceils that express CD200 and lack expression of HLA-O, and isolating said cells from other cells to form a cell population
  • a cell population is produced by selecting placental eeltsfoat (a) adhere to a substrate, and (b) express CP73, Cpioi, and Ci>2 ⁇ N3 ⁇ 4 and isolating said cells from other cells to form a cell population
  • a cell population is produced by jderrtifyihg placental cells that express CD73, CD 105, and CD200, end isolating said cells from other cells to form a celt population, to Mother embodiment a cell population is produced
  • a cell population is produced try selecting placental cells that express CD200 and OCT-4. and isolating said celts from other cells to form a cell population.
  • a cell population is prodded by selecting placental cells that (a) adhere to a substrate, (t>) express CD73 and CD 105; and (C) facilitate the formation of One or more emfrfyoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for toe formation of an erobtyOid-like body; and isolating said ceils from other cells to form a cell population, to another embodiment, a cell population is produced by selecting placental cells that express CD73 and CD 105, and facilitate the formation of one or more embtyoid-Iike bodies in a population of placental cells comprising said stem cell when said population is culttoed under conditions that allow for the formation of an embiyoid-iike body ,
  • a cell population is prodneed by selecting placental ceils that (a) adhere to a substrate, and (b) express CD73 and CD 105, and lack expression of HIA ⁇ G; apd isolating said cells from other cells to fa a cell population;
  • a cell population is produced by selecting placental cells teat express CD73 and CD105 and lack expression of HLA-G, and isolating said cells from other celb to form a celt population, to mother embodiment
  • foe method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, (b) express OCT-4, and (c) facilitate foe formation of one or more emtoyoid-likc bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for foe formation of an embryoid-like body; and isolating said cells from other cells to form a cell population.
  • a cell population is produced by selecting placental cells that express OCT-4, and facilitate the formation of one or more embryoid-tike bodies in a population Of placental cells comprising said stem cell when said population is cultured under conditions that allow for the formation of an embryoid-like body, and isolating said cells from other cells to form a cell population.
  • a celtpopuMon is produced by selecting placental cells that (a) adhere to a substrate, and (h) express CD 10 and CD 105, an d do notexpress CD34; and isolating said cells froth other cells to form a cell population
  • a cell population i$ produced by selecting placental cells that express CD10 and CD 105, and do not express CD34.
  • a cell population is produced by selecting placental cells that (a) adhere to a substrate, and (b) express CD10, CD105, and CD200, and do not express CD34; and isolating said cells from otoer cells to form a cell population,
  • a cell population is produced by selecting placental cells that (a) adhere to a substrate, and (b
  • a cell population is produced by selecting placental cells that express CD10, CD90, CDI05 and CO2O0, and do not express CD34 and CD45, and isolating said cells from other cells to form a cell peculation.
  • selection of the isolated cell populations can additionally comprise selecting placental cells that express AB ⁇ 1 ⁇ 2 (a placenta-specific ABC transporter protein; see, e.g., Ailikmets ef al., Cancer Res.58(23):5337-9 (1998)), the method can also comprise selecting cells exhibiting at least one characteristic specific to, e,g. ? a mesenchymal stem cell, for example, expression of CD44, expression of CD90, or expression of a combination of the foregoing.
  • AB ⁇ 1 ⁇ 2 a placenta-specific ABC transporter protein
  • the substrate cab be any surihee on which culture and/or selection Of cells, e g., isolated ptaCentot cells, can be accomplished.
  • the substrate is plastic, e.g., tissue culture dish or multiwell plate plastic.
  • Tissue culture plastic can be coated with a biOmolecule, e.g., laminin or fibifoeectto.
  • Cells e.g., isolated placental cells, pan be selected for a placental cell population by any means known to the art of cell selection.
  • cells can be selected using an antibody or antibodies to one of more cell surface markers, for example, in flow cytometry o* FACS. Selection can be accomplished using antibodies in conjunction with magnetic -beads.
  • Antibodies that are specific for certain stem cell-related markets ate known in the art Fry example, antibodies to OCT-4 (Abeam, Cambridge, MA), 00200 (Abeam), HLA-G (Abeam), CD73 (BD Biosciences Pharmingen, San Diego, CA>, CD105 (Abeam; Bioftesign international, Bacp, MS), etc.
  • Antibodies to other markers are also available commercially, e.g., 0P34, CD38 and CD4S are available from, e.g., StemCell Technologies or BioDesigo International.
  • the isolated placental cell populations can comprise placental cells that ate not stem cells, or cells that are not placental cells.
  • the isolated cell populations comprising placental derived adherent cells described herein can comprise a second cell type, e,g., placental cells that are not placental derived adherent cells, or, e.g., cells that arc not placental cells.
  • an isolated population of placental derived adherent cells can comprise, e.g., can be combined with, a population df a second type of cells, wherein said second type of Ceil are, e.g., embryonic stem cells, blood cells (e.g., placental blOod, placental blood cells, umbilical cord blood, umbilical cord blood cells, peripheral blood, peripheral blood cells, nucleated cells from placental blood, umbilical cord blood, or peripheral blood, and die like), stem celts isolated Shorn blood (e.g., stem celts isolated from placental blood, umbilical cord blood or peripheral blood), nucleated cells from placental perfusate, e.g., total nucleated cells from placental perfusate; umbilical cord stem cells, populations of blood-derived nucleated cells, bone marrow-derived mesenchymal stromal cells, boue marrow-derived mesenchymal stem cells, bone marrow-derived
  • a population of cells comprising placental derived adherent cells comprises placental stem cells or stem cells from umbilical cord.
  • the second type of cell is blood or blood cells
  • erythrocytes have been removed from the population of cells.
  • the second type of cell is a hematopoietic stem celt
  • Such hematopoietic stem cells can be, for example* contented within unprocessed placental. ttmbUicalcord blood or peripheral blood; to total nucleated cells from plftoestitol blood, umbilical cord bipod or peripheral blood; m an isolated population of CD344 ⁇ cells from placental blood, Umbilical potd blood or peripheral blood; to unprocessed tome marrow; to total nucleated cells from bone marrow; to an isolated population ofCD34* cells from bone marrow, or die like,
  • an isolated population of placental derived adherent cells is combined with a plurality of adult otptogehitoi cells from the vaseular system to various embodiments, the cells are endothelial cells, endothelial progenitor cells, myocytes, cardiomyocytes, pericytes, angidblasts, myoblasts Or cardiomyoblasts.
  • the second cell type is a non-embryonic cell type manipulated in culture in order m express markets Of pltir ipoteneyand functions associated with embiyonicstem cells
  • either or both of the placental derived adherent cells and cells of a second type are autologous, or am allogeneic, to an intended recipient of the cells.
  • the composition comprises placental derived adherent cells, and embryonic stem cells, to another specific embodiment, the composition comprises placental derived adherent celts and mesenchymal stromal or stem cells, e,g., bone marrow-derived mesenchymal stromal or stem cells. In another specific embodiment, the composition comprises bone matron-derived hematopoietic stem cells. In another specific embodiment, the composition comprises placenta!
  • tire Composition comprises placental derived adherent cells and somatic stem cells, to a more specific embodiment, said somatic stem cell 1 ⁇ 4 a neural stem cell, a hepatic stem celt, a pancreatic stem cell, an endothelial stem cell, a cardiac stem cell, or a muscle stem cell.
  • the second type of cells compri se about, at toast, or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells in said population.
  • the PDAC in Said composition comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of cells in said composition, to other specific embodiments, (fie placental derived adherent cells ccanpiise about, at least, or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% ofeel!s in said population.
  • Cells in an isolated population Of placental derived adherent cells can be combined with a plurality of cells of another type, e,g > , with a populatibn of stem cells, in a ratio of about 100, 000,000: 1 , 50,000,000: 1, 20,000,000: 1 , 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1,5:1,2:1, 1:1: 1:2; 1:5; 1:10; 1:100; 1:200: 1:500; 1:1,000; 1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000; 1 :50, 000,000; or about 1:100,000,000, comparing numbers of total nucleated cells in each population.
  • a population Of the placental ceils described herein, e.g., the PDACs described above, are combined with osteogenic placental adherent celts (OPACs), eg, the OPACs described in Patent Application No, 12/546, 5S6, ftied August 24, 2009, entitled “Methods and Compositions for Treatment Of Bone Defects With Placental Stem Ceils,” dr combined widi amraon-derived angiogenic cells (AMDACsX the AMDACs described in
  • placental cells described herein can be combined with any physiologicaily-acceptable or ntedically-acceptable compound, composition Or device tor use in tite methods and compositions described herein.
  • Compositions useful in toe methods of treatment provided herein can comprise any one or more of die placental ceils described herein.
  • die composition is a pharmaceuticafly-acceptable composition, e > g., a Composition comprising placental cells in a pharmaceutically-acceptable carrier.
  • a Composition comprising tiie isolated placental cells additionally comprises a matrix, e.g., a decellularized matrix or a synthetic matrix, to another specific embodiment, said matrix is a three-dimensional scaffold.
  • said matrix comprises collage**, gelatin, lathimti, fibrooectiu, pectin, oroifhme, or vitronectin, in andflier ore specific embodiment, the maw is an amniotic membrane or an amniotic membrmie-derived biomaterial,
  • said matrix comprises anextiacelhilar membrane protein.
  • said matrix comprises a synthetic compound.
  • said matrix comprises a bioactive compound, in atiother specific embodiment said bioactive compound 1 ⁇ 2 a growth factor, cytokine, antibody, or organic molecule of less than 5,000 daltons.
  • composition nsefitl in the methods of treatment provided herein comprises medium conditioned by any of the foregoing placental cells, or any of the foregoing placental cell populations,
  • Isolated placental cell populations useful m the methods and compositions described herein can be preserved for example, cryopreserved for later use. Methods for cryopreservaiion of cells, such as stem cells, are well known in the art.
  • Isolated placental cell populations cm be prepared in a form that is easily adnunistrable to an individual, e.g., an isolated placental cell population that is contained within a container that is suitable for medical use, Such a container can he, for example, a syringe, sterile plastic bag, flask, jar, or other container from which the isolated placental cell population can be easily dispensed.
  • She container can be a blood bag or other plastic, medically-acceptablebag suitable fbr the intravenous administration ofaliquid to a recipient, the container, in certain embodiments, is one that allows for cryopreseryafibn of the combined cell population.
  • the cryopreserved isolated placental cell population can comprise isolated placental cel! derived from a single donor, or from multiple donors;
  • the isolated placental cell population can be completely HLA-matched to an intended recipient, m partially m completely HLArmismatched,
  • isolated placental cells can be used m the methods and described herein in the form of a composition comprising a tissue culture plastic-adherent placental cell population in « Container.
  • the isolated placental cells are cryopreserved.
  • the container is a bag, flask, or jar, Itt another specific embodiment, said bag is a sterile plastic bag; In another specific embodiment* said bag is imiteble for * allows or facilitates intravenous administration pfsaid isolated placental cell population, e.g., by iniravenous infusion.
  • the bag can comprise multiple lumens or compartments that are interconnected to allow mixing of the isolated placental cells and one or more other solutions, e g., a drug, prior to, or during, adramistration.
  • the composition comprises one ortnore compounds that facilitate cryopteservatioft of the combined cell population, th another specific einbodunetit; said isolated placental cell population is contained wttitin a physiologicafly-accep table aqueous solution.
  • said physiolOgically-acceptable aqueous solution is a 0,9% NaCl solution
  • said isolated placental cell population comprises placental cells that are MLA-matched to a recipient of said cell population.
  • said combined cell population comprises placental cells that are at least partially H LA- mismatched to a recipient of said cell population, in another specific embodimen t, said isolated placental cells are derived from a plurality of donors.
  • the isolated placental ceils in the container are isolated
  • CD 10T, CD34-, CD105+ placental cells wherein said cells have been crybpreserved, and are contained within a container.
  • saidCDlOK CD34 ⁇ , CDlOS-t placental cells are also CD200t.
  • said CDJO 1 * * CD34-, CD105f , CD2QCH placental cells am also CD4S - pr Ct>9(hh
  • said CDl(H% CD34-, CD 105*, CD20CN- placental cells am also CD45- and CD9tH * .
  • CDl(MyCDlO$* placental cells are additionally one or mom of CD 13*, CD29+, CD33+, CD38-, CD44*, CD45-, CD54+, CD62E-, CD62L-, CD62P-, SH3+ (CD73+), SH4* ⁇ C&73*>, CD80- CD86-, CD90?, $112* (CD105+), CD106/VCAM*, CDU7-, CD144/VE- cadhertndim, CD184/CXCR4- CD200+ CD 133-, OCT-4+, SSEA3 ⁇ , SSEA4-, ABC-p+, KDR- ⁇ VEOFR2-), HCA-A,B,C*, HLA ⁇ DP,DQ,DR- HLA-G-, or Progmmmed Death-1 Ligand (PDLI )+, or any combination thereof, h another specific embodiment, the CD34TM, CDlOf, CD105+ placental cells aiead
  • the above-referenced isolated placenta! cells ait isolated CD200+, HLA-G- placental cells, wherein saidcellshave been cryopreserved, and are contained within «container.
  • the isolated placental cells are CD73+, CD10S+, CD20ti+ cells that have been cryopreserved ⁇ aod are contained within a container.
  • the isolated placental cells are CD20CH , OCT-4+ stem cells that have been cryopreserved, and are contained within a container,
  • the isolated placental cells are CD73+ CD105+ cells that have been cryopreserved, and an?
  • the isolated placental cells facilitate the formation of one Or more embryoid-like bodies when cultmred with a population of placental cellstrader conditions that allow for the formation of embryoid-tifce bodies
  • the isolated placental cells are CD73+, €D105+, HLA-O- cells that have been cryopreserved, and are contained within a container.
  • the isolated placental cells are OCT-4+ placental cells that have been cryopreserved, and are contained within a container, and wherein said cells facilitate the formation of one or more embtyoid-ltke bodies when cultured with a peculation of placental cells under conditions that allow for the formation ofembryoid-like bodies.
  • the above-referenced isolated placental cells are placental stem cells or placenta) multipotenr cells that are CD34-, CD10+ and CD1Q5+ 1 ⁇ 4 detected by flow cytometry (e.g., PDACs),
  • foe isolated CD34-i CD 10*, CDI05+ placental cells have the potential to differentiate info cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype
  • foe isolated CD34-, GD10+, CD1G5+ placental cells are additionally 002004.
  • foe isolated C034-, CDJ04, CD105+ placental cells am additionally CD5KH- Or CD45-, as detected by flow cytometiy.
  • foe isolated CD34-, €010+, CD10R placental cells are additionally CD9CH or CD45»:, as detected by flow cytometiy.
  • CD 1054, CD200+ placental cells are additionally CD90+ or CD4S-, as detected by flow cytometry.
  • CD34-, CD ICJMvCDlOS*, CD200+ cells are additionally CD90+ and CD4S- as detected by flow cytometry.
  • foe CD34-, CDJ 0+, CD 105* CD29G+, CD904, CD45- bells ate additionally CD80- and CD86-, as detected by flow cytometry;
  • CDI05+ bells are additionally one ormoie ofCD29+, CD3SK Cp44*, CD54+ ⁇ Cb$ ⁇ K CPSfK SH34 or SH4+V
  • the cells are additionally CD44+.
  • the celis are additionally one or mote of Cm 17TM, 03133-, KDR- (VEGFR2-), HLA-A,B.C+ , flLA ⁇ Ol ⁇ D&DR n and/or PDLH, f 601971
  • said container is a bag.
  • said container comprises about, at least, or at most i x 10* said isolated placenta! cells, 5x !0* said isolated placental cells, l x to 7 said isolated placentalcelfs, 5X 10 ? said isolated placental cells,lx 10* saidtsolated placental cells, 5 x 10 s said isolated placental cells, l x I O 9 said isolated placental cells, 5 x 10
  • said isolated placenta! cells, 1 x 10** said isolated placental cells, or 1 x 10 id said isolated placental celts toother specific embodiments of any ofthe foregoing cryopreserved populations, said isolated placental celts have been passaged about, at least, or no more than 5 times, no mom than IQ times, no more titan 15 times, or no more than 20 times in another specific embodiment Of any of the foregoing cryopreserved isolated placental cells, said isolated placental celts have been expanded within said container
  • a single unir dose Of placental derived adherent cells can comprise, m various embodiments, about, at least, or no more than 1 x lO 3 , 3 x 10 3 , 5 x 10 3 , 1 x 10 4 , 3 x 10 4 , 5 x !0 4 , 1 x lO 5 , 3X lp*, 5 X lO-Yl x 10* 3X 10 6 , 5 x l ⁇ , 1 X W, 3X 10T, 5 x !Q 7 , 1 x 10 s , 3 x 10 s , 5 x 10 ⁇ I x I0 9 , 5 x lO 9 , or 1 x 10 m placental cells.
  • a single unit dose of placental derived adherent celts can comprise between l x IQ 3 to 3 x l Q ⁇ 3 x 10* to 5 x IQ 3 , 5 x 10* to I x iQ 4 , 1 x 10* to 3 x 10 4 , 3 x l0*to 5 x 10 4 , 5 x 10* to 1 x 10 $ , I x 10 5 to 3 x 10 s , 3 x 10 s to 5X 10*,.5 x IQ $ to 1 x K 1 x 10 6 to 3 x ⁇ 6 , 3 x 10* to 5 x 10 6 , 5 x lO 6 to 1 x IQ 7 , 1 x l0 7 to 3 x 10 7 , 3 x 10 7 to 5 x IQ 7 , 5 x IQ 7 to 1 x 10 s , 1 x 10 s to 3 x 10, 3 x ⁇ 0 to 5 x 10 4
  • the pharmaceutical compositions provided herein comprises populations of placental derived adherent cells, that comprise 50% viable cells or more (that is, at least 50% of the cells in tile population ate functional or living).
  • populations of placental derived adherent cells that comprise 50% viable cells or more (that is, at least 50% of the cells in tile population ate functional or living).
  • at least 60% of the cells in the population are viable.
  • at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable.
  • compositions for use in vivo can be formulated into pharmaceutical compositions for use in vivo, e,g., in the methods of treatment provided herein,
  • Such pharmaceutical compositions comprise a population of isolated placental cells, of a population of cells comprising isolated placental cells, in a pharmaceutically-acceptable carrier ⁇ e.g. f a saline Solution or other accented physiologicatly-aceeptable solution for in vivo administration.
  • Pharmaceutical compositions comprising the isolated placental cells described herein can comprise any, or atty combination, Of the isolated placental cell populations, or isolated placental cells, described elsewhere herein.
  • the pharmaceutical compositions can comprise fetal, maternal, or both fetal and maternal isolated placental cells.
  • the pharmaceutical compositions provided herein can further comprise isolated plhcental cells obtained 1 ⁇ 2m a single individual or placenta, or from a plurality of individuals or placenme:
  • a single unit dose of placental derived adherent cells can comprise about, at least, or no mote titan 1 x IG ⁇ 3 ⁇ 10 5 , 5 x 10*, 1 x 10*, 3 x 10 4 , 5 x 10 4 , 1 x 10 s , 3 x 10 5 , 5 x 10 s , 1 x 10* 3 x 10 6 , 5 x 10*, 1 x 10 ?
  • the pharmaceutical compositions provided herein ate administered tb a subject having diabetic peripheral neuropathy once.
  • the pharmaceutical compositions provided herein ate administered to a subject having diabetic peripheral neuropathy bti multiple occasions, e,g., twice, three titties, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times. Intervals between dosages can be weekly, bi-weekly, monthly, bi-monthly or yearly. Intervals can also be irregular.
  • Doses ofplaceniat stem ceils administered according to such regimens include, but are not limited to, ⁇ X 10 7 , 3 x 10*, 5 X 10 3 , 1 X IO 4 , 3 X 10*. 5 x 10 4 , 1 x IO 5 , 3 X 10*, 5 x 10 s , 1 ⁇ 10 6 , 3 X 10* 5 x M* I x !io 7 , 3 x 10 ⁇ > 5 x JO 7 , !
  • the dose ofplacental stem cells in aphanuaceutreal composition is I ⁇ 10 3 placental stem cells, 1» another specific embodiment* the dose Of placental stent cells in a pharmaceutical composition is 3 X 10 ⁇ placental stem cells.
  • ftie dose of placental stem cells in a pharmaceutical composition ' is 3 x W placenta! stem cells» to another specific embodiment, die dose ofplacental stem cells in a pharmaceutical composition is 3 x 10 s placental stem cells, to another specific embodiment, the dose ofplacental stem ceils 1 ⁇ 2 a pharmaceutical composition is 1 x 10 ® placental stem cells, to another specific embodiment, die dose ofplacental stem cells in a pharmaceutical composition is 3 x 10* placental stem cells. In another specific embodiment, toe dose of placental stem cells in apharmaceutical composition is3 x lO 7 placental stem cells.
  • a pharmaceutical composition comprising placental Stem cells (e.g, ⁇ CD1 ⁇ K CD105+, cmm, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy once as a single dose.
  • a pharmaceutical composition comprising placental stem cells e.g;, CDS Or, CD105+, CD20Q*, CD34- placental stem cells
  • a pharmaceutical composition comprising placental stem cells is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a Second dose about ! week later
  • a pharmaceutical composition comprising placental stem cells (&.g consult 0>!CH% CD!
  • 05*, CD200*, CD34- placental stem cells is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about 1 week late- and a third dose about one week after that (i.e., about two weeks after die initial administration).
  • Doses of placental stem cells administered according to such regimens include, bat are not limited to, 1 x IQ 3 , 3 x 1 ⁇ *, 5 x 10 3 ,
  • tile dose of placeniaistem cells in a pharmaceutical composition is 1 x 10 j placental stem cells, in another specific embodiment, the dose of placental stem cells m a pharmaceutical cotoposition is 3 x 10 s placental stem cells, tat another specific embodiment, the dose of placenta!
  • stem cells in a pharmaceutical composition is 3 x ⁇ 4 placental stem cells.
  • toe dose of placental stem cells in a pharmacetitical composition is 3 x lO 5 placental stem cells.
  • the dose of placental stem cells m a pharmaceutical composition is I x 10 s placemal stem cells.
  • tiie dose of placental stem cells in a pharmaceutical composition is 3 x 19* placental stem cells.
  • toe dose ofptacental srem cells m a pbamiacetdic ⁇ composition is 3 x tO 7 placental stem cells.
  • a pharmacetitical composition comprising placental stem cells (e.g., CDlO*, CD10S ⁇ . CDKXH-, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about l month later (e.g., about 27, 28, 29, 30 > 31, 32, or 33 days after the initial dose).
  • placental stem cells e.g., CDlO*, CD10S ⁇ . CDKXH-, CD34- placental stem cells
  • a pharmaceutical composition comprising placental stem cells (e.g., CD ICHh, CblOS ⁇ CD200+, CD34- placental stem celts) isadministered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about I month later and a third dose about one month after that (i,edeem about two months after toe initial administration, e.g;, on of about day 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 following the initial administration).
  • placental stem cells e.g., CD ICHh, CblOS ⁇ CD200+, CD34- placental stem celts
  • Doses of placental stem cells administered according to such regimens include, but are not limited to, 1 x lO 3 , 3 x 10 3 , 5 k 10 3 , l x Iff, 3 k lO 4 , 5 x 10 4 , 1 x 19 s , 3 x IQ 5 , 5 k 10*, 1 x 10*.3 x IP 6 , 5 x 10*, 1 x IQ 7 3 X JO 7 , 5 X tO 7 , 1 x 10 *, 3 x 10 s , 5 x 10 s , 1 x IQ 9 , 5 x 10 s , or 1 x 10’* placental cells or between 1 x 10 3 to 3 x lO 1 , 3 x 10 s to $ x 10* 5 x iO’to 1 x 10 4 , l x 10 4 to 3 x 10 4 , 3 x 10 4 to 5 x 10*, -5
  • toe dose of placental stem cells in a pharmaceutical composition is l x 19 3 placental stem dells.
  • the dose of placental stem Cells in a pharmaceutical composition is 3 x 10 3 placental stem cells.
  • the dote of placental stem cells in a pharmaceutical composition is 3x 19* placental stem cells.
  • the dose of placental stem cells in «pharmaceutical composition is 3 x lO 5 placental stem cdljs.
  • the dose ofplacental stem cells in a pharmaceutical composition is 1 K lO 6 placental stem cells, in another specific embodiment, the dose Of placental stem cells in a pharmaceutical composition is 3 X, 1 Of placental stem cells. In another specific embodiment, the dose of placental stem cells in a pjmrmaceutical composition is 3 x i0 7 placental stem celts.
  • compositions provided herein Comprise populations of cells that comprise 50% viable cells or titoire (that is, at least 50% Of the cel ls in the population am functional dr living). Preferably, at least 60% of tire cells m the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the ceils in the population ip die pharmaceutical composition are viable;
  • compositions provided herein can comprise one Or more compounds that* «,$.* facilitate engrafiment (e.g., anti ⁇ T-cCll receptor antibodies, an immunostippressant, or die like); stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, plasmalyte, and the like.
  • compounds that* «,$.* facilitate engrafiment
  • stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, plasmalyte, and the like.
  • the pharmaceutical composition comprises about 1% to 1.5% ESA and about 2,5% dextran.
  • the pharmaceutical «imposition comprises from about ⁇ x 106 cells per milliliter to about 2 x 10* cells per milliliter m a solution comprising 5 % HSA and 10% dextran, optionally comprising an immunosuppressant, e.g., cyclosporine A at, e.g., 10 ntg3 ⁇ 4.
  • the pharmaceutical composition e.g., «solution
  • the pharmaceutical composition comprises between about 1.5 x 10 6 celts per milliliter to about 3.75 x 10* cells per milliliter.
  • the pharmaceutical composition comprises between about 1 x !0* cells/mL to about 50 x 10 s ce!is/mL, about 1 x l0 6 cells/mL to about 40 x 10* cells/mL, about I x 10* cells/mL to about 30 x 10* cells/mL, about ) x 10* celfs/mL to about 20 x 10* cells/mL, about I x !O 6 cetis/mL to about 15 x 10 s cells/mL, or about 1 X JO* cells/mL to about 10x 10* cells/mt .
  • the pharmaceutical composition comprises ho visible ⁇ ell clomps (U., no tiraerocell clumps), or substantially nosuch visible clumps,:
  • ho visible ⁇ ell clomps U., no tiraerocell clumps
  • substantially nosuch visible clumps As used hereby ‘"macro cbticlbmps ⁇ means an aggregation of cells visible without magnification, e.g., visible to the naked eye, and generally refers to a cell aggregation larger than about 150 microns
  • the pharmaceutical composition comprises about 2.5%, 3.0%, 3.5%, 4,0%, 4.5%, 5.0%, 5.5%, 60%, 6.5%, 7.0%, 7.5% 8,0%, 8.5%, 9,0%, 9.5% or 10% dextran, e:g., dextran-40, in a specific embodiment, said composition comprises about 7>5% to about 9% dextran-40.
  • said composition comprise about 5-5 % dextrrm-40, in certain embodiments, the pharmaceutical composition comprises from about 1% to abemt 15% human seniUi albumin (HSA). In specific embodiments, the pharmaceutical composition comprises about 1%, 2%, 3%, 4%, 5%, 65, 75, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% HSA. In a specific embodiment, said cells have been cryopreserved and thawed.
  • said Cells have been filtered through a 70 pM to 100 ⁇ filter, in another specific embodiment, said composition comprises no visible ceil clumps, in another specific embodiment* said composition comprises fewer than about 200 ceil clomps per 106 cells, wherein said cell clumps are visible only under a mierpseope, e.g., a, tight microscope. In another specific embodiment, said composition comprises fewer than about 1 $0 cell clumps per 10* cells, wherein said cell clumps are visible only under a microscope, e.g., a tight mibrbscope.. ⁇ another specific embodiment, said composition comprises fewer than about 100 celt clamps per 10* celts, wherein said cell clumps ate visible only under a microscope, e.g., a light microscope.
  • the pharmaceutical composition comprises about 1.0
  • a pbarmaceuttcal composition comprising placental stem cells provided herein comprises about 5.75% dextrin 40, about 10% human serum albumin, and about 2.5% DMSO.
  • the pharmaceutical composition comprises a plurality of qe3 ⁇ 4 e,g tennis a plurality of Isolated placental cells in a solution comprising 10% dextran-40, wherein fee pharmaceutical composition comprises between about 1.0 ⁇ 0.3 x 10* cells per milliliter to about 5.0 ,*1.5 x 10* cells per milliliter, and wherein said composition comprises so cell clumps visible with the unaided eye (i.e., comprises no macro cell clumps).
  • the pharmaceutical composition comprises between abbot 15 x 10* cells per milliliter to about 3,75 x 10* cells per milliliter, to a specific embodiment said cells have been cryopreserved and thawed.
  • said cells have been filtered through a 7QpM to 100 ⁇ filter.
  • said composition comprises fewer than about 200 micro cell clumps (that is, cell clumps visible only with magnification) per 10* cells.
  • me pharmaceutical composition comprises fewer than about 150 micro cell chimps per 1# cells. tn another specific embodiment, the pharmaceutical
  • composition comprises fewer titan about 100 micro cell dumps per JO* cells, to another specific embodiment the pharmaceutical composition comprises less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, : 4%, 3%, or 2% DMSO, or less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0,3%, 0,2%, or 0.1% DM$0.
  • compositions comprising cells, wherein said compositions ate produced by one of the methods disclosed herein.
  • the pharmaceutical compo&Hion comprises cells, wherein the pliatmaceutical composition is produced by a mtihod comprising filtering a solution comprising placental cells, e,g Berry placental stem cells or placental multipoient cells, to form a filtered cell-containing solution; diluting the filleted cell-contaimiig solution with a first solution to about I to 50 x 10 s , 1 to 40 x 10 6 , 1 to 30 x 10* t to 20 x 10*, I to 15 ⁇ x I0*, or 1 to 10 x i0*ed&pe?
  • milliliter * e.g., prior to cryopreservation; and diluting the resulting filtered cell-containing solution with a second solution comprising dextrah, but not comprising humatt serum albumin (USA) to produce said composition.
  • said diluting is to no more than about 15 x 10* cells per milliliter.
  • said diluting is to no mote than about 10 * 3 x 10 6 ceils per milliliter.
  • said diluting is to no more than about 7,5 x 10* cells per mi lliliter.
  • filtration is optional.
  • the filtered cell-containing solution, prior to the dilution comprises less than about 10 * 3 x 19* ceils per milliliter. filtration is optional. In ptiier certain embodiments, if the filtered cell-containing solution, prior to the dilution, comprises less than about 7.5 x 10 s cells per milliliter, filtration is optional
  • the cells are ciyopreserved between said diintmg with a first dilution solution and said diluting with said second dilution solution.
  • the first dilution solution comprises dextran mad HSA.
  • the dextran in the first dilution solution or second dilutiou solution can be dextrah of any molecular weight, e,g » , d&ttran having a molecular weight of from about 1 O kDa to about 150 kDa.
  • said dextran in said first dilution solution or said second solution is about 15%, 3.0%, 3,5%, 4,0%, 4.5%, 5.0%, 53%, 6,0%.6.5%, 7,0%, 7.5% 80%, 8,5%,: 9,0%, 9.5% or 10% dextran.
  • toe dextran in said first dilution solution or said second dilution solution is dextran-40.
  • toe dextran in said first dilution solution mid said second dilution solution is dextian-40
  • said dextran-40 in said first dilution solution is 5.0% dextran-40.
  • said dextran-40 in said first dilution solution is 5.5% dextran-40; In another specific embodiment » said dextran-40 in said second dilution solution is 10% dextrau-40.
  • said HSA in send solution comprising HSA is 1 to 15 % HSA.
  • said HSA in mid solution comprising HSA is about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 101 ⁇ 4, 11%, 12%, 13%, 14% or 15 % HSA.
  • said HSA to said Solution comprising HSA is 19% HSA.
  • said First dilution solution comprises HSA- to another specific embodiment, said HSA in said first dilution solution is 10% HSA. to another specific embodiment, said first dilution solution comprises a ccyoprotectant. to another specific embodiment, said ctyopratectant is DM$Q * to another specific embodiment, said dextran-40 in said second dilution solution is about 10% dextran-40. In another specific embodiment, said composition comprising cells comprises about 7,5% to about 9% dextran. to another Specific embodiment, the pharmaceutical composition comprises from about 1.0 *0.3 x 1 (C ecils per milliliter to about 5.0 *1.5 x 10* cells per milliliter.
  • the pharmaceutical composition comprises from about 1,5 x l ⁇ f cells per milliliter to about 3.75 x W celts per milliliter.
  • toe pharmaceutical composition is made by a method comprising (a) filtering a cell-containing solution comprising placental cells, e.g., placental stem cells or placental multipotent cells, prior to cryopreservation to produce s filtered cell-containing solution; (b) cryopreservmg the cells in the filtered cell-containing solution at about 1 to 50 x 10», 1 to 40 x If, 1 to 30 x W, I to 20 x W, 1 to 15 x 10*, or 1 to 10 x W cells per milliliter; (c) thawing the cells; and (d) diluting toe filtered cell-containing solution about 1 il to about 1 ill
  • step (b) are ctyopreserved at about to . ⁇ £ ⁇ .3 x lO 6 cells per totHititeir.
  • the cells in step (b> are cryopreserved in a solution comprising about 5% to about ip% dextran-40 and MBA. in certamembodiments, said diluting in step (b) is to no more tban about 15 x . 10* cells per milliliter.
  • the pharmaceutical composition is made by a method comprising', (a) suspending placental cells, placental stem coils or placental retiltipotent cells, in a 5.5% dextran-40 solution that comprises 10% HSA to form a cell-containing solution; (b) filtering the cell-containing solution through a 70 pM filter, (c) dilating the cell-containing solution with a solution comprising 5.5% dextran-40, 10% HSA, and 5% DMSO to about 1 to 50 X 10 6 , 1 to 40 x 10 s , I to30x 10 6 , 1 to20x 10 s , 1 to J5x t0 6 ,or 1 td lOx 10* cells per milliliter, (d) cryopreservtng the cells; (e) thawing to* cells; and (f) diluting the cell-containing solution 1 : 1 to 1 : 11 (v/v) with 10% dextran-40.
  • said diluting to step(c) is to no more tban about 15 x 10 6 cells per milliliter. In certain embodiments, said diluting in step (c) is to no more than about 10 *3 x 10* cells/mL. In certain embodiments, said diluting in step (c) is to no mote than about 7.5 x 10* cells/mL.
  • the compositiitto comprising cells is made by a method comprising: (a) centrifuging a plurality of cells to collect the cells; (b) resuspending the cells to 5, 5% dexti3 ⁇ 4» «40; (c) cenuifuging the cells to collect toe cells; (d) resuspending toe cells in a 5,5% dextran-40 solution Aat comprises 10% HSA; (e) filtering the cells through a 70 ⁇ filter, (i) diluting the cells in 5:5% dextran-40, 10% HSA, and 5% DMSO to about 1 to 50 * lot I to 4OX 10 6 , 1 to 30x 10*, 1 toTOx IQ 6 , I to l5.x ld 6 , or 1 to 10 x 10* cells per milliliter; ⁇ g) cryopresetvmg the cells; (hj thawing the cells; and (i) diluting toe cells 1 :1 to 1
  • said diluting to step (f) is to no more than about 15 x lO 6 cells pertoftlititer. In certain embodiments, said diluting in step (f) is to no more than about 10 ⁇ -3 x l O 6 cells/mL. In certain embodiments, said diluting in step (f) is to no more than about 7.5 x 10* Cells/mL. In other certain embodiments, if toe number of cells is less than about 10 db 3 x 10* celts per miflilhet, filtration is optional.
  • compositions e.g., pharmaceutical compositions comprising the isolated piacental cells. described herein can comprise any of the isolated placental cells described herein. t3 ⁇ 4i02!5I Other injectable formulations, suitable tor the administration of cellular produces, maybeused.
  • the pharmaceutical composition comprises isolated placental cells that are substantially, orconrpletely* tton-materaal in origin, that is, have the fetal genotype; e.g., at least about 90%, 95%, 98%, 99% or about 100% are non-matemal in origin.
  • a pharmaceutical composition comprises » population of isolated placental cells that ate CD200+ and HLA-G-; CD73+, CDIG5+, and CD20&+; CD2(KH ⁇ and OCT-4+; CD73+, CD !
  • a pharmaceutical composition comprises a population of isolated placental cells that am CDitK CD1GS+ and CD34-; CDl ⁇ H , CD 105+, 00200+ and CD34-; CDltK CDl05t, CD200+, CD34 " and at least one of CD90+ or CD45-; CDKH, CD90+, CD105+, CD20Q+, CD34- andCD45 ⁇ ; CD10+, CD90+, CD1G5+, CD200+, €D34- and CD45-; CD20(H and HLA-G-; CD73+, CD105+ and CD200+; CD2004 ⁇ and OCMn CD73+, CD1054 ⁇ and HLAdS-s CD73+ and CD 105+ and facilitate the formation Of One or more embryoid-like bodies m a popplfttion of placental cells comprising said isolated placental cells when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like body; OCT
  • the pharmaceutical composition additionally comprises a stem cell that is not obtained from a placenta.
  • a stem cell that is not obtained from a placenta.
  • Isolated placental calls in the compositions, e g., pharmaceutical compositions, provided herein, can comprise placental cells derived from a single donor, or from multiple douora Hie isolated placental cells can be completely HL A-matched fo an mlerided tecipient, or partially ojrcompletely HLAmmmatched.
  • compositions comprising matrices, hydrogels, scaffolds, and the like that comprise a placental cell, m a population of isolatedplacental cells.
  • Such compositions can he used in the place Of, or in addition to, cells in liquid suspension.
  • the isolated placental ceils described hereto can be seeded onto a natural matrix, e.g,, a placentalbiomaterial such as an amniotic membrane material.
  • a placentalbiomaterial such as an amniotic membrane material.
  • an amntotic membrane material can be, e.g., amniotic membrane dissected directly from a mammalian placenta; fixed or heat-treated amniotic membrane, substantially dry (i.e., ⁇ 20% H20) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like.
  • Preferred placental biomatetials cm which isolated placental cejls can be seeded are described in Hariri, U.S. Application Publication No. 2004/0048796, the disclosure of which is incorporated herein by reference to 1 ⁇ 2 entirety ,
  • the isolated placental cells described herein can be suspended in a hydrogel solution suitable for. e.g., injection; Suitable hydrogels for such compositions include self- assembling peptides, such as RAD 16.
  • a hydrogel solution comprising the cells can be allowed to harden, for instance in a mold, to form a matrix having cells dispersed therein for implantation. Isolated placental cells to such a matrix can also be cultured so that the cells are mjtotically expanded prior to implantation.
  • the hydrogel is.
  • Hydrogel-forming nwterials include polysaccharides such as alginate and salts thereof peptides, poiyphosphaztnes, and polyacrylates, which toe crossimked ioriically, or block polymers such is polyethylene oxide-polypropylene glycol block copolymers which are crosslinkedby temperature or pH, respectively, in some embodiments, the hydrogel or matrix is biodegradable.
  • die formulation comprises an in situ polymerisable gel (see,> e.g., U S. Patent Application Publication 2002/0022676, toe disclosure of which is incorporated herein by reference in its entirety; Ansefe et al sociall Control Release, 78(1-3): 199- 209 (2002); Wang et al., Biomaterials, 24 ⁇ 22):3969-80 (2003).
  • the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof.
  • aqueous solutions such as water, buffered salt solutions, or aqueous alcohol solutions
  • polymers having acidic side groups that can be reacted With cations are poiyiphospha ⁇ nes), p «ty(acfyiic acidaX poly(mefoacryHc acids), copolymers of acrylic acid and methacrylic acid, polyfvinyl acetate), andsulfonated polymers, such as sulfonated polystyrene.
  • Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used.
  • acidic groups arc carboxylic add groups, sulfonic add groups, halogenated (preferably fiuorihated) alcohol groups, phenolic QH groups, and acidic QH groups.
  • the matrix is a felt, which can be composed of a multifilament yam made from a bioabsorbable material, e.g., PGA, PLA, PGL copolymers or blends, or hyaluronic acid.
  • the yarn is thade into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling, to another preferred embodiment the cells of the invention am seeded onto foam scaffolds feat aiay be composite structures.
  • fee three-dimensional framework may he molded into a useful shape, such as a spetific structure in the body to be repaired, replaced, or augmented.
  • Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VlCPYL, Ethicon, toe., Somerville, N J.). foams, composed of. e.g via poly(e-caprolactone)/poly(glycolic acid) (RCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilization (see, e.g., O.S. Pat No. 6,355,699), can also be used as scaffolds.
  • PGA/PLA synthetic absorbable copolymer of glycolic and lactic acids
  • RCL/PGA poly(e-caprolactone)/poly(glycolic acid) copolymer
  • the isolated placental cells described herein or cd-cultures thereof can be seeded onto a feree-dimensiotial framework or scaffold and implanted in vivo.
  • a framewodi can be implanted in combination with any one or more growth fectors, ce3 ⁇ 4 drugs or other components feat, e g., stimtilate tissue formation.
  • Nonwoven mats can be formed using fibers comprised Of a synthetic absorbable copolymer oigiycOlicand Me acids (e.g., PdA/PLA) (VICRYL, Efoicoh, foe,, Somerville, NIL).
  • Foams composed of, e ⁇ g., poly(e-caprolactoneXpo]y(jgIycolic acid) (PCE/PGA) copolymer, formed by processes such asffeeze-drying, or lyophilizatiou (see, e.g., O.S. Pat. No, 6355,699), can also be Used as scaffolds.
  • isolated placental cells can be seeded onto, or contacted with, a felt, which edit be, e.g., competed of a muttifilameiit yam made froro a btoabsorbable materia! such as JP ⁇ 3 A, PLA, PCL copolymers or blends, or hyaluronic acid.
  • a muttifilameiit yam made froro a btoabsorbable materia! such as JP ⁇ 3 A, PLA, PCL copolymers or blends, or hyaluronic acid.
  • the isolated placental cells provided herein can, in another embodiment, be seeded onto foam scaffolds that may be composite structures.
  • foam scaffolds can be molded into a useful shape, such as that of a portion of a specific structure in the body to be repaired, replaced oraugmented, lit some embodiments, the ftameworkis treated, e.g., with 0.1 M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of foe cells in order toenhatice cell attachment.
  • External surfaces of a matrix may be modified to improve foe attachment or growth of cells and differentiation of tissue, such as by plasma- coating the matrix, or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosammogiycans fog. , heparin sulfate, chonfooitin*4-stilfate, chondroitin-ti-sulfate, dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/or other materials Such as. but not limited to; gelatin, alginates, agar, agarose, and plant gums, and foe like.
  • proteins e.g., collagens, elastic fibers, reticular fibers
  • glycoproteins glycoproteins
  • glycosammogiycans fog. e.g., heparin sulfate, chonfooitin*4-stilfate, chondroitin-t
  • the scaffold comprises, or is treated with, materials that render it noo-fotombogenjc, These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition.
  • these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as EPT.FE, and: segmented polyurefoaneurea silicones, such as PURSPANTM (The Polymer Technology Group, Inc., Berkeley,
  • the scaffold can also comprise anti-thrombotic agents such as heparin; the scaffolds can also be seated lo alter foe surface charge fog. * coating with plasma) prior to seeding with holated placentai cells,
  • the placental cells fog., PDACs provided herein can also be seeded onto, or contacted with, a physiologically-accq)table ceramic material including, but not limited tb, mono-. dk tri-, elpha-trkbetartri-, atid teira-calcium phosphate, hydroxyapatite, fluoro ⁇ tites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BlOGLASS$, and mixtures thereof.
  • a physiologically-accqtable ceramic material including, but not limited tb, mono-. dk tri-, elpha-trkbetartri-, atid teira-calcium phosphate, hydroxyapatite, fluoro ⁇ tites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BlOGLASS$, and mixture
  • Porous biocompatible eeiamic materials currently commerciallyavaiWle include SURG1BGNES (CsnMedica Coip., Canada), ENDOBON® (Merck Biomaterial France, Fmee), CEROS® (Mathys, AO, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOSTM (DePuy, Inc., Raynham, MA) and V!TOSS®, RHAKQSSTM aiid CORTOSS® (Orthovita, Malvern, Pa).
  • the framework can be a mixture, blend or composite of natural and/or synthetic materials,
  • the isolated placental cells are seeded onto, or contacted with, a suitable scaEold at about 0.5 x IQ* to about 8x 10* eefts/mL
  • #602311 Provided herein ate methods of treating an individual having a disease, disorder or condition, wheteiu the disease, disorder or condition is caused by , or 1 ⁇ 2 associated with, an inappropriate or undesirable immune response, e.g., a disease, disorder or condition that can be treated beneficially by immunosuppression, comprising administering to the individual placental stem cells, to a specific embodiment, the amount is an amount sufficient to delectably suppress an immune response in the individual
  • ah immune response can be, e g., proliferation of T cells in an MLR or regression assay performed using T cells from the individual #602321 Ah individual having a disease, disorder or condition associated with or caused by an inappropriate or undesirable immune response, e.g., an individual having, or at risk of developing multiple sclerosis; a person having, or at risk of developing, an inflammatory bowel disease, e g., Crohn's disease or ulcerative colitis; a person having or at risk of developing graft* Vc3 ⁇ 4sus-host
  • the individual can be treated immediately after diagnosis, or within 1 , 2, 3, 4, 5, 6 days of diagnosis, or within l, 2, 3, 4, 5, 6, 7, 8, 9, I D, 15, 20, 25, 30, 35, 40, 45, 50 or mote weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after diagnosis;
  • the individual can be treated puce, or multiple times during the clinical course of die disease.
  • the individual can be treated, as appropriate, during an acute attack, during remission, or during a chronic degenerative phase, f 002331
  • the placenta stem cel ls usefiil h die treatment of suc h a disease, disorder or
  • the placental stem cells express CD200 and HLA-G; express CD73, CD105, and CD200, express CD200 and OCT4; express €D73, GD105 and HLA-G; express CD73 and CPIOS, and facilitate the formation of one of more embryoid-like bodies in a population of placental cells when said population 1 ⁇ 4 cultured under conditions that allow for rite formation of embryoid- Hke bodies; Or expressCK’TA, and (c) fedlitate the formation of one ortnire embryoid-Bke bodies to a population of placental cells wheat said population is cultured under conditions that allow 3 ⁇ 4 the formation of embryoid-like bodies; or any combination of the foregoing.
  • the placental stem cells are CDWs, CD 1054, CD20Q+, CD34* 1 placental stem cells.
  • the placental stem cells are CD! 174.
  • the individual is administered » dose Of about 300 million placental stem cells. Dosage, however, can vary according to toe individual's physical characteristics, e.g., weight, add can range from 1 miilion to 10 billion placental stem cells per dose, preferably between 10 million and I billion per dose, or between 100 million and 50 million placental stem cells per dose.
  • the administration is preferably intravenous, but can be by any medicalty-acceptable route for the administration of live cells, e.g., paremerally, subcutaneously, intramuscularly, intraperitoneally, intraocularly, and the like. Id one embodiment, the placental stem cells are from a cell bank.
  • a dose of placental stem cells e.g., from amnion, amnion/chorion, chorion or umbilical cord, is contained within a blood bag or similar bag, suitable tor bolus injection or administration by catheter, 1002351
  • a method of treating an indi vidual having a disease, disorder or condition, wherein the disease, disorder or condiuon is caused by, or is associated with, an inappropriate or undesirable immune response, e.g., a disease, disorder or condition that can be treated beneficially by immunosuppression comprising administering to the ifldi vidiml euhute medium that baa been conditioned placental stem cells, in an amount sufficient to detectably suppres s an immune response in the indi vidual.
  • Such an immune response can be, eg., proliferation of T dells in an MLR or regression assay performed using T cells from the individual.
  • ⁇ 88236 ⁇ Placental stem ceils or umbilical cord stem celts, or medium conditioned by placental stem celUt or umbilical cord stem ce3 ⁇ 4 can be administered 1 ⁇ 2 a single dose, or in multiple doses. Where placental stem cells are administered in multiple doses, the doses can be part of a therapeutic regimen designed to relieve one .
  • disease, disorder or condition wherein the disease, disorder or condition is caused by* or is associated with, aft inappropriate or undesirable imtntitte response, or can be part df a long-term therapeutic regimen designed to prevent, or lessen the severity, of a chronic course of such a disease, disorder Or condition.
  • j802371 In another aspect, provided herein is a method of treating an individual haying multiple sclerosis, or a symptom associated with multiple sclerosis, comprising administering to die individual a plurality Of placental stem cells, or medium conditioned by placental stem ce3 ⁇ 4 in an amount and for a time sufficient to detectably modulate, e.g., suppress an immune response in the individual.
  • j 80238 j Multiplesclerosis (MS) is a chrome, recurrent inflammatory disease of the central
  • the disease results in injury to the myelfo sheaths surrounding CNS and PNS axons, oligodendrocytes; and the nerve cells themselves.
  • the disease is mediated by autoreactive T celts, particularly CD4+ T cells, that proliferate, cross foe hloodforain barrier, mid enter die CNS under the influence of cellular adhesion molecules and pro-mflammatoiy cytokines, bubble symptoms Of MS include sensory disturbances in the limbs, optic nerve dysfunction, pyramidal tract dysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia, and
  • RRMS relapsing/temitting MS
  • SPMS secondary progressive MS
  • FffMS primary progressive MS
  • PRMS progressi ve/relapsing MS
  • PRMS progressi ve/relapsing MS
  • Person® having MS are generally evaluated usihga motor skii jts assessment, optionallywitb an MRL For example » one motor skills assessment, the expanded disability stams scale, scores gradations h an affected individual's abilities, as follows:
  • MS has also been assessed by magnetic resonance imaging, which cab detect neural lesions associated with MS (e.g., new lesions, enhancing lesions, or combined unique active lesions):
  • a method of treating an individual having MS e.g., and individual who has been diagnosed with MS, comprising administering to the individual a plurality of placental stem cells sufficient to delectably suppress an immune response 3 ⁇ 4 the individual.
  • the MS is relapStog/itmftting MS.
  • the MS is secondary progressive MS.
  • the MS is prhnary progressive MS.
  • the MS is progressive/relapsing MS.
  • the administering delectably improves one or more symptoms of MS in the individual.
  • the symptom is, e.g., one or more of a sensory disturbance in the limbs, an optic nerve dysfunction, a pyramidal tmctdysfunction, a bladder dysfunction, a bowel dysfunction, a sexual dysfunction, ataxia, or diplopia.
  • saidadministering results in an improvement on the BDSS scale of at least one half point
  • said administering results in the maintenance of function, according to at least one MS scoring system, over the course of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months
  • said administering results in an improvement on the BOSS scale of at least one point
  • said administering results in an improvement on the EDSS scale of at least two points.
  • said administering results in a detectable improvement on a multiple sclerosis assessment scale or bn an MRl
  • the individual can be treated, as appropriate, duringunacute attack, during remission, of during a chronic degenerative phase.
  • the placental stem cells ate administered to a female having MS * post-partuiu, to maintain the state of remission or reduced occurrence of relapse experienced during pregnancy,
  • the therapeutic agent is a glucocorticoid.
  • the ghicocorticoid b adrenocorticotropic hormone (ACTH), methy Iprednisotoite , or dexaroethasone is a glucocorticoid.
  • the ghicocorticoid b adrenocorticotropic hormone (ACTH) methy Iprednisotoite , or dexaroethasone.
  • the therapeutic agent is ah immimomodulatory or immtmosuppressive agent.
  • the immunomodulatory' or immunosuppressive agent is ⁇ - 1 a, IFN- 1 b, gliairiamer acetate, cyclophosphamide, methotrexate, azathioprine, cl*dribine, cyclosporine m mitoxmitrane.
  • the therapeutic agent is intravenous immunoglobulin, plasma exchange, Or sulfasalazine.
  • the individual is administered any combination of the foregoing therapeutic agents.
  • placental stem cells, placental stem cell populations, and/or compositions comprising placental stem cells or placental stem cell populations are used to treat an individual bating, or at risk of developing, inflammatory bowel disease (IBD), e.g., Crohn’s disease or Ulcerative colitis.
  • IBD inflammatory bowel disease
  • a method of treating an individual having inflammatory bowel disease, or a symptom associated with inflammatory bowel disease comprising administering to the individual a plurality of placental stem cells, or medium conditioned by placental stem cells, in an amount and for a time sufficient to delectably modulate, e.g., suppress an immune response to die individual.
  • the IBD is Crohn’s disease, sometimes referred to as ileitis or enteritis.
  • Crohn’s disease is a chronic disorder tliat causes itffiammation of the digestive tract (also referred to as toe gastrointestinal, or 01, tract). Crohn’s disease can affect any part of toe 0l tract, firbm mouth to anus, but most comm only affects the lower part of the small intestine, referred to as the ileum. Five types of Crohn’s disease are known. Gastroduodenal Crohn’s disease afflicts toe stomach and duodenum (the faintest portionoffee «hall intestine).
  • lejunoileitis is Crohn’s diseaseof the jejunum, the longest portion of the small intestine.
  • Ileitis isCrohn's disease of the ileum, the lower portion of die small Intestine, Ileocolitis, die most common form ofCrohn'sdjsease, effects the ileum and colon.
  • FinalIy, Crohn’s cotitis ⁇ Gfanulomatous colitis affects the colon, and is distinguished from ulcerative colitis in that in Crohn’s colitis, them are often areas of healthy tissue between ureas tif diseased tissue, andCrohn3 ⁇ 4cotiti$ can involve only the colon, without involving the rectum.
  • Crohn’s disease is thought to arise from inappropriate reaction of the body’s irnmime system to antigens in the Gl tract, including, e,g., food, beneficial bacteria, etc., resulting man accumulation of white blood cells in the lining of the intestines.
  • Inflammation associated with Crohti’s disease has also been attributed to the action of fee cytokine tumor nebosis factor (TNF «a).
  • Ulcerative colitis is a disease that causes inflammation and sores (u!cers)in the lining of the rectum and/or colon. Ulcers fbim where inflammation has killed the cells that usually line the colon; the ulcers typically subsequently bleed and produce pus. When inflammation occurs in the rectum and lower part Of the colon, the disease 1 ⁇ 2 referred to as ulcerative proctitis. If the entire colon is affected, fee disease is called pancolttis.
  • ulcerative colitis Symptoms of ulcerative colitis include, but are not limited to, abdominal paid, bloody diarrhea, fevers, nausea, abdominal cramps, anemia, fatigue, weight loss, loss Of appetite, rectal bleeding, loss of bodily fluids and nutrients, skin lesions, joint pain, and growth failure (in children). Ulcerative colitis can also cause complications stick as inflammation of the eye, liver disease, and osteoporosis.
  • a method of treating an individual having an inflammatory' bowel disease comprising administering a therapeutically effective amount of placental stem Cells to said individual, wherein said therapeutically effective amount is an amount feat results in a detectable improvement in at least one symptom of said inflammatory bowel disease (IBD).
  • IBD inflammatory bowel disease
  • the IBD is Crohn’s disease.
  • said Crohn’s disease is gastroduodenal Crohn's disease, jefenoileitis, ileitis, ileocolitis, or Crohn’S colitis.
  • said symptom is a symptom of Crohn’s disease»
  • said symptom of Crohn’s disease is inflammation and swelling of a part of the GI tract, abdominal pain, frequent emptying of the bowel, and/or diarrhea.
  • said symptom of Crohn’s disease is rectal bleeding, anemia, weight loss, arthritis, ikin problems, fever, toiCkening of the intestinal wall, formation of scar tissue in the intestines, formation of sores or ulcers in the intestine, development of cate or more fistulas in the intestinal wall , deve lopment of one or mote fissures in the antis, development of nutritional deficiencies (e.g.* deficiencies in one or more of proteins, calories, vitamins), development of kidney stones, dev elopment of gallstones, of diseases of the liver or biliary system.
  • nutritional deficiencies e.g.* deficiencies in one or more of proteins, calories, vitamins
  • said Ulcerative colitis is Ulcerative proctitis, pancohtis, limited colitis or distal colitis
  • said symptom is a symptom of ulcerative colitis:
  • said symptom is abdominal pain, bloody diarrhea, fivers, nausea, abdominal cramps, anemia, fatigue, weight loss, loss of appetite, rectal bleeding, loss of bodily fluids and nutrients, Skin lesions, joint pain, and growth failure.
  • the symptom is osteoporosis, eye inflammation, or liver disease.
  • said individual to whom placental stem cells are administered 1 ⁇ 4 additionally administered one or more of a second therapy, wherein said second therapy comprises an anti-inflammatory agent, steroid, immune suppressor, and/or an antibiotic;
  • anti-inflammatory drugs useful in the treatment of Crohn’s disease or ulcerative colitis include, but are not limited to, mesalamine, 5- ASA (5 -aminosalicylic acid) agents (e.g., ASACOIS (mesalamine, delayed-retease), PIPENTUM (Osalazine), PENTASA® (tnesalamme controlled-release)), sulfasalazine (a combination of 5-ASA and sutfapyridine), antiinflammatory antibodies (e.g.
  • Infliximab (REM!CADES)
  • steroids useful to tihe treatment of Crohn’s disease or ulcerative colitis
  • examples of steroids useful to tihe treatment of Crohn’s disease or ulcerative colitis include, but are not limited to, cortisone, hydrocortisone, predisone, methylprednesone, and tile tike.
  • the dosage of steroid ts first delivered in a relatively large dose, followed by smaller dosages as inflammation subsides.
  • immune suppressors useful in the treatment of Crohn’s disease include, but ate nor limited to, cyclosporine A, 6-mercaptopurine or azathioprine.
  • Any antibiotic can be used 1 ⁇ 2 toe treatment of Crohn's disease, including, e.g, fKapiciltin* sulfonatnide. cephalospottim teiracyctine, and/or metronidazole EXAMPLES
  • Opti-MEM medium 125ul of Opti-MEM medium ⁇ 6.25ug of Cas9 nuclease (&A36493, ThermoFisherXnvitrogen), 1.2ug of sgRNA, and 12.5ul ofCas9 Plit$ reage «t (WCMAX00008, ThermoFtsher Invttrogen) in Tube t.
  • Immediately add solution from Tube! to Tube 2 X then mix well, incubate complex for 5-10 mins at RT. Add 250ul of complex into each well. Incubate cells for 2 days at 37 ?C. After mcabatkm, remove APPL-001 culture medium and feed cells with fresh culture mediant;
  • Standard Curve Reconstitute the TF Standard with 0,8 ml reagent grade water to generate a solution of 250 pM Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (250 pM) 1 :2 with Sample Dihient to FOduce 125, 62.5, 31.25, 15.63, 7.81 and 3.906 pM. Sample Diluent serves as the zero standard (0 pM). Freshly prepare the desired volume of Assay Mix by combining the following reagents.
  • the values represent the volumes for one reaction: Assay Diluent 50 FV$ iOfoL, atiti PX ifofoL Add 7Q pLofthe Assay Mix to each well Add 10 ⁇ > oftissue Factor Standard or sample to each well Mix gently. Incubate at 37°C for 30 minutes in a humid incubator to avoid drying out the wells. Add 20 pL ofFXa Substrate to each well and mix gently. Cover We3 ⁇ 4 with a sealing tape and incubate at 3 ?*C and read absotbances oh a tnicroplate reader tit * wavelength of 405 ntn every 5 minutes ft» 35 minutes. Incubate micfbplate at 37°C after eachreading PGE2 potency assay
  • PCR conditions were 1 min at 95°C (1 *), followed by 15 s at 95°C, 15 s at 55° € and 1 min at 72°C (30*).
  • the 40ul of PCR products were sent for sattger sequencing in Genewiz. Sequencing data were uploaded to https;//tide.nldn
  • TF triggers coagulation protease cascade and activates both FIX to FIXa and FX to FXa. This leads to the formation of low anmuntsof thrombin, which activates the cofactors FV and FVIll and cause the safety issues in patient treatment.
  • BM-MSC has low TF expression level:
  • TF activity of APPL-TFKO is compared with control (non -edited) and human BM ⁇ MSd. In FIGS; 3A-3B, activity was reduced from control cells in both cell lines by >90%. Both whole cells and cell lysates showed remarkable reduction of TF activity; TF activi ty level in whole cells of APPL- TFKO was as low as that in BM-M SC.
  • FIGS; 4A-4C demonstrated that both TFKO-B and TFKO-C werea maintained in 974 and 995 as cells expand (torn pi to p6.
  • TFKO-B was stably maintained in .981 from pl to p4, however, not maintained at p5 mid p6.
  • TP activity showed that sgRNA ⁇ C mediated TFKO activity reduetionretained at p6, and it was as low as that in MSC, Observed increased relative TF activity of pd ceils were at both $81 and 974 atp6,
  • ⁇ 602581 AFPLs were characterized by a panel ofmarkers
  • CD lO-t-ZCDl 05+/CD20CH-/CD34 " To characterize APPL-TFKO celt lines, we checked this nommal phencrfype markers by FACS for 981 , 974, and 995 lines.
  • FIG; 6 showed that CD34 expression level is ⁇ 1%, CDlO+ and CD 1054 level is >99%, and CD200 is >90% for all three cell lines. SgRNA*B/C did hot show difference in nominal genotype.
  • ⁇ 60266 ⁇ APPL-AE and -AL are standard APPL cell lines established ih CCT.
  • APPL-AE and ⁇ AL ⁇ TFKQ cdl lines with sgRNA-B.
  • IT expression level of these TFKO cell lines by FACS mid TIDE assay, to FACS, TFKO by gRNA-b achieves >95% in both standard cell lines (FIGS. 8A-8B),
  • TFKO confirmation by TIDE assay IWtrn to further confirm TFKO in APPL eeU lines tested by FACS, we checked editing efficiency of by sanger sequencing and TIDE assay in 981, 97% AE and AL.
  • A is DNA gel imaging for product for senger sequencing, which cowed the editing region
  • FIG, 9 demonstrated ihatsgRN A*B led to a high editing efficiency (>85%) in ail four cell lines, which was consistent with FACS results. Editing efficiency is lower ( ⁇ 50%) by sgRNA-C m 974 cells With low R2 value dne to low quality Of FOR samples;
  • FIG. 12 showed that CD34 expression level is ⁇ 1%, CDJtft- and CD105+ level is >99%, and CD200 is >90% for both AE/AL-CTR and-ko B cells (FIO.
  • FIGS. I3A-13B showed that TFKO in 98 i ⁇ 974, AE and At cells inhibited boteCD4 and CDS T cell proliferation.
  • M2 macrophages with antiinflammatory properties ate associated with wound healing and tissue repair4
  • APPL cells have immune regulatory properties including an anti-inflammatory activity, beneflting the wound heating process.
  • Fig. demonstrated that Control and TFKO cells increased the expression of M 1 marker CD206 and M2 marker CD80 in dOnor AL, 98 i , and 974, and increased CD8G only in donor AE.
  • FIG. 15 demonstrated tee decreased TF activity in four APPL-TFKO cells compared!» control cells, which was consistent with TF expression data. Phenotype analysis showed all four TFKO lines maintains APPL phenotype (CD34- €D105+GD20(HCD10 ⁇ ).
  • PGE2 secretion potency of APPL-TFKO (00269)
  • P6E2 potency of control and TFKO tit 981 and 974 lines We have examined whether lTKOtiy sgRNA-B would affect PGE2 secretion in AE and AL, APPL cells were stimulated with IL- l b. After 24 hours, secreted PGE2 was measured by ELISA.
  • PGE2 secretion level was normalized by XTT metabolic activity, PGE2 activity assay showed that AL demonstrated similar PGE2 secretion profiles between the control and TFKO-B.
  • Afe-TP&O increased PGE2 secretion by 2 folds compared to control cells (FIG, 16),
  • MSCs Mesenchymal stenv'stromal cells
  • exosomes which are a diameter of 40-150 nm membrane-bound vesicles with functions as mediators of cell-cell communication.
  • MSC-derived exosomes contam cytokines, growth factors, bioactive lipids, RNAs, and proteinsS.
  • Increasing evidence suggests that MSC-derived exosomes might represent a novel cell-free therapy with compelling advantages over parent MSCs such as no ride of tumor formation and lower immunogeuietty.
  • cytokine, HGF* was highly detected in both APPL 981 control and TFKO cell lines; cytokines, including MCP-J , RANTES; and lL-8, had a low to moderate level.
  • CRISPR/Cas enables a targeted modification of DNA sequences.
  • easeUnd efficient use one limitation is the potential occurrence of associated off-target effects.
  • whole genome sequencing (WGS) is tiie moat common detection method allowing the identification of off-target effects in a less restricted way6.
  • WGS t we fan a qttiOW and easier test, RNA-seqttencing, to analyze and compare the transcriptome of gene expression patterns in between APPL parental control cells and APPL- TFKO cells,
  • IF127 is a hydrophobic mifocbdndrial protein composed of l22 amino acid [5).
  • the OAS2 protein is a well-known innate immune activated antiviral enzyme catalyzing synthesis of 2 # -S' -oligoadenyiate for RNase Lactivation and inhibition of viral propagation.
  • OAS2 expression was upregulated m ail conditions and correlations between metby!ation and expression were seen in psoriasis and tongue SCO.
  • Schipl interaction parmer Merlm belongs to die family of ERMproteins (Ezrin, Radix in and Moesin) thatregulation not only as linkers between the cell membrane and the cortical cytoskeleton, but also as active modulators of the aetm eytoskeieton and membrane receptors tii the subcortical areas bf cellextensious.
  • ERMproteins Ezrin, Radix in and Moesin
  • Colony-stimulating foctor 3 is a cytokine used clinically tot promoting the production and release of hpiie maifow-derived hematopoietic stem cells and granulocyte [9].
  • CSF3 has been shown to affect additional aspects of the innate and adaptive immune Systems.
  • CSF3 inhibitioft increased T eeti infiltration, and decreased neoplasm devbfopment by 75% in die AOM/BSS model of CRC development [14].
  • CXCLl C-X-C Motif Chdmokine Ligand 1
  • CXCLl acts as a chemoattractant for several immune cells, especially neutrophils.
  • Diseases associated with CXCLl include ICaposi Sarcoma and Bacterial Meningitis. Among its related pathways are OCRS Pathway In Macrophages and PEDF Induced Signaling.
  • ⁇ 00282 ⁇ TFKO in AE were upregtiJated PCDHGB4 by 14475 folds, ACTAl by 333 folds, SNX22 by 46 folds, CD163 by 24.8 folds and were downregulated CIQTNF5 by 100 folds and AGAP5 by 30 folds.
  • PCDHGB4 Protocadberin Gamma Subfamily B, 4 belongs to the hugest mammalian Subgroup of the cadherin superfamily ofhomophilic cell-adhesion proteins. Diseases associated with PCDHGB4 include Papillary Craniopharyngioma.
  • ACTA 1 100284 ⁇ Actin Alpha 1 , Skeletal Muscle (ACTA 1 ) provides instructions for making a protein called skeletal alpha (a)-actrtu which is pari of the actin protein family. Skeletal ct-actin plays an important role in skeletal muscles Diseases associated with ACTAl include Myopathy, Scapuiohumeropcroneal and Ndmaline Myopathy 3. Among its related pathwaysare RhoGDi Pathway and PAK Pathway.
  • SNX22 (Sorting Mtutm 22).
  • the protein encoded by this gene is a sorting nexin that is found in the cytoplasm, where it interacts with membrane-bound phosphatldylinoshol 3- phosphate.
  • the encoded protein may play a role in intracellular trafficking.
  • Diseases associated with SNX22 include Osteogenesis Imperfecta, Type lx and Hemochromatosis Type 2. (902861 duster of Dverenttatiou $63 fC£>lB> Is foe high aBnity scavenger receptor for the hemoglobm-haptoglobin complex and in the absence of haptoglobin- with lower aBnityr for hemoglobin alone.
  • CD 163 functions as innate immiine sensor ibr gram-positive and gram-negative bacteria.
  • a soluble form of the receptor exists in plasma, and cerebrospinal fhhd.
  • sCDl 63 is upregulated in a large range Of itiflaminatoty diseases including liver cirrhosis, type 2 diabetes, macrophage activation syndrome, Gaucher's disease, sepsis, Hi V infection, rbeumatoid artbritis and Hodgkin lymphoma.
  • sCDI 63 is also upregulated id cerebrospinal fluid after subarachnoid Imemorrhage.
  • C1QTNF5 tumor necrosis factor related protein 5
  • 1602891 TFKO in 981 were upregulated FAM187A by 3553 folds, BIVM-ERCC5 by 22 folds, and were down-regulated TMEFF1 by 5000 folds, EXOSC6 by 25 folds, and NPB by 1 $ folds.
  • FAM187A Family With Sequence Similarity 187 Member A
  • Diseases associated with FAM187A include Ciliary Dyskinesia, Primary, $7 and Chromosome 17Q12 Duplication Syndrome.
  • B1VM-ERCC5 is related tp single-stranded DNA binding andnuclease activity.
  • Diseases associated with BIVM-ERCC5 include Cerebrooculofocfoskeletal Syndrome 3 and Xeroderma Pigmentosum, Comple Group G. Among its related pathways are Nucleotide excision repair.
  • TMEFFl is a novel transmembr ane protein, composed of two follisatin domains and a unique epidermal growth factor (EGF)-like region. These structural domains suggest a role for TMEFFl in tbe regulation of growth factor signaling either as a ligand precursor , a membrane-bound receptor or as a binding protein for growth factors, TMEFFl may inhibit NODAL and BMP signaling duringneural patterning and may be a tutnotsuppressor in brafo cancers.
  • EXOSCO ExoSonte complex exonuclease Mills constitutes one of the subunits of the multisubunit particle called tile exosome complex, which mediates mRN A degradation.
  • NBP Neuropeptide B
  • Nf*R Niemann-Pick Disease
  • Type B sud Gallbiadder Papillomatosis X- Linked Nephrolithiasis Type 1 and Planter Wart * (602941 Neuropeptide B (NPB) may regulate feeding, pain perception, and stress in rodents
  • Diseases associated with Nf*R include Niemann-Pick Disease, Type B sud Gallbiadder Papillomatosis.

Abstract

Provided herein are improved placental-derived tissue culture plastic-adherent cells, e.g. cells which have been genetically modified to reduce tissue factor expression, and uses thereof.

Description

APPL CELLS IMPROVED PLACENTAL-DERIVED ADHERENT CELLS AND
METHODS OF THEIR USE
0001 This application claims priority to U.S. Provisional Patent Application Nos.
63/070,034, filed August 25, 2020, 63/076,647, filed September 10, 2020, and 63/202,481, filed June 14, 2021 , the contents of each of which are incorporated heroin in their entities.
1 FIELD
0002 Provided herein ate improved placental-derived tissue culture plastic-adherent cells, e.g, colls which have been genetically modified to reduce tissue factor expression, and uses thereof.
L BACKGROUND
0003 Allogenic Pluripotent placenta cells ( APPL) are a culture expanded, adherent
MSG like population isolated by mechanical and enzymatic digestion of human placental tissue. APPL exitibit a phenotype of CD34 -, CD10 +, CD105 +, and CD200 GE2 secretion is used as potency assay for APPL in Diabetic Foot Ulcer (DFU) with and without Peripheral Arterial Disease (PAD) and Diabetic Peripheral Neuropathy (DPN), Like their bone marrow counterparts, APPL possess potent immune suppressive properties and exert their effects on T lymphocytes through a multitude of mechanisms that include cell contact mediated interactions and through the modulation of secreted soluble factors. APPL have been shown to inhibit both the proliferation and cytokine production of T lymphocytes* as well as modulate T cell differentiation!,
0004 Tissue factor (TF), also called CD142, is a protein encoded by the F3 gene, present in subendothelial tissue and leukocytes. TF plays an essential role in coagulation and hemostasis. The TF :FV!la complex is the key initiator of foe coagulation protease cascade and activates both FIX to PlXa and FX to FXa. this leads to the formation of low amounts of thrombin* which activates the tofactots PV and FVHL2,3 Different MSG products display vary ing levels of highly procoagalamtissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (1BM1R). MSCs derived from perinatal tissue (PT) has higher expression of TF than those from adipose tissue (AT) and bone marrow (DM). Our product, Human placenta-deri ved cells (PDA-001), was tested in patients with multiple sclerosis and patients with active rheumatoid arthritis, showing safety concerns for coaugulation and hemostasis due to high expression Of TF oh PDA-001. There exists, therefore, an unmet need for placental stem cells which retain desired biological properties but with reduced TF expression and or activity and an unproved safety profile,
3. SUMMARY
0005 The FDA has recommended reduced TF expression without compromising the potency of the targeted PDA-001 biological activity. Therefore, previous tear» in CCT mitigated TF expression by using shRNA-mediated TF knockdown (KD). Historical data have shown that TFKD reduced TF expression by 60-70%, as well as TF activity by 60-70%. TFKD also decreasedin vitro clotting and coagulation by 60-80%. Functional studies have demonstrated that TFKD did not affect APPL suppression of T cell proliferation and activation: However, they found that TFKD abrogated APPL activity on M1 downregulation and abolished PDAC efficacy in rat neuritis (inflammatory pain model). In order to reduce TF expression without affecting APPL biological activities, tins study is aiming to generate new generation APPL with TF knockout (TFKO) by using CRIPSR gate editing technology.
0006 Experiments were carried out to generate the stable APPL-TFKO cell lines via sgRNAZCas9 Ribonucleoprotein (RHP) system. The TFKO efficiency was examined by TIDE assay and FACS. The cells with stable and highly efficient TFKO were cryopreserved throughout cell expanding up to passage d. During the passage, APPL-TFKO were Characterized by TF expression, TF activity, nominal phenotype markers, PGE2 may, and secretome analysts. Furthermore, the potency of APPL-TFKO biological activity were tested by in vitro bead T-Cell reaction (BTR) assay and in vivo mouse EAE model. The results demonstrated that TrueGuide™ Synthetic sgRNA CR1SPR688420_SGM (thermoFisher) resulted in > 90% TFKO and KO were able to be stably maintained along cell passaged. We also found that TFKO significantly reduced its enzymatic activity by > 89%. Additionally* die expression of APPL nomi nal phenotype markers, CD l0+/CD20tH/CDl 05+/CD34- were not altered in APPL-TFKO. Secretome analysis compared secreted fhetore between APPt-CTR and APPL-TFKO, and revealed that TFKO did not affect secretome of APPL. As for the functional studies, APPL-TFKO suppressed the proliferation and activation of T cells in vitro and showed the in vivo efficacy of mouse EAE model
160071 The present invention provides a population of placental stem cells which are
CD 10+, CD34-,CD105+ CD200+,and CD142-, In some embodiments, the population of placental stem cells ate additionally negative for one or more markers selected from the group consisting of CD117, CD45, and CD133, In some embodiments, rite population of placental stent cells am additionally negative for CD117. some embodiments, the population of placental stent cells am additionally Negative for CD45. In some embodiments, depopulation Of placental stem cells are additionally negative for CD133.
0009 in some embodiments, the population of placental stem cells are additionally positive for One of mom markers selected from the group consisting of CD44, CD90, OcH, and HLA-G. In some embodiments, the population of placental stera cells are additionally positive for CD44. In some embodiments, the population of placental stem cells are additionally positive for CD90. In some embodiments, tire population of placental stem cells are additionally positive fat Oct-4. In some embodiments, the population of placental stem cells are additionally positive for HLA-G.
In some embodiments, the population of placental stem cells are genetically modified. In some embodiments, the genetic modification is a tissue factor (CDl 42, F3 gene) knockout- In some embodiments, the genetic modification is a CRISPR tissue factor (CD142, F3 gene) knockout.
00011 In some embodiments, the cells induce increased anti-inflammatory macrophage activity relative to CD142+ PDAC cells, in some embodiments, the increased anti-mflammatory activity comprises increased expression of CDSO on M2 macrophage. In some embodiments, the cells induce increased pro-inflammatory maerophageactivity relative to CDl 42+ PDAC cells. In some embodiments, the increased anti-inflammatory activity comprises increased expression of CD206 on Ml macrophages. 00012 In some embodiments, the cells have increased antt-inflammatory properties relative' to CD142+ PDAC cells, in some embodiments, the cells express increased an increased marker of activity under inflammatory conditions relative to CD 142+ PDAC cells. In some embodiments, the increased marker of activity is PGE2. In some embodiments, the inflammatory condition is IL-lb stimulation.
00013 The present invention also provides a pharmaceutical composition comprising the population of placenta) stem celts of tire invention and a pharmaceutically acceptable earner. 00014 Hie present invention also provides a method of treating a disease or condition in a subject in need thereof, the method comprismg the step of administering to the subject a therapeutically effective amount of the population of placental stem cells of the invention or the pharmaceutical composition of the invention, so as to thereby treat the subject In some embodiments, the subject is a mammal. In some embodiments, the subject is a human, administration is intravenous. In some embodiments, the administration is local. In some embodiments, the administration is intramuscular;
00015 M some embodiments, the subject has an autoimmune disease. In some embodiments, the autoimmune disease is multiple sclerosis. In some embodiments, the subject has an inflammatory bowel disease. In some embodiments, the inflammatory bowel disease is Crohn's disease;
00016 The present invention also provides & method of suppressing an tmmune response comprising contacting a plurality of immune cells with a population of placental stem ceils of the invention for & time sufficient fear said placental stem cells to delectably suppress an immune response. In some embodiments, the plurality of immune cells are T cells or NK (natural killer) cells; In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD84 T cells, til some embodiments, the contacting is performed in vitro. In some embodiments, the contacting is performed in vivo. In some embodimems, the contacting is performed in a mammalian subject In some embodiments, the mammalian subject is a human subject.
BRIEF DESCRIPTION OF THE FIGURES 0017 FIGS. 1A - 1B show sgRNAs-mediated TFKO in 981 and 995 at different seeding density by FACS and TIDE.
0018 FIGS.2A - 2B show reproducibility of TFKO by sgRNA B or C in 981, 974, and
§95 tines.
0019 FIGS. 3A - 3B show that IHO reduced TF activity in both whole cells and ceil
0020 FIGS.4A - 4C show that TF expression and activity was maintained as APFL- TFKO expanded to p6.
0021 FIG.5 sbows that TFKO did not cbange protiferation of APPL tines.
0022 FIG.6 shows that TFKO did not alter nominal phenotype of APPL. 0023 FIG. shows PGE2 secretion of APPL lines,
0024 FIGS. SA - 8B show TFKO by sgRNA-B in AB and AL.
0025 FIG, 9 shows TFKQ confirmation by TIDE assay,
0026 FIG, 10 shows the viability of AE arid AL
0027 FIG. 11 shows TF activity of 981, 974, AE, and AL at late passage.
0028 FIG. 12 shows the nominal phenotypes of AE and AL
0029 FIGS. 13 Α- 13B show that suppression of T-cell proliferation was maintained in
APPL-TFKO.
FIG. 14 shows M1/M2 macrophage polirization induced by APPL-CTR/TFKO.
0030 FIG, 0 shows TF expression and phenotype analysis of 4 ΈΑΕ study cell lines.
0031 FIG. i 6 shows PGE2 secretion pf APPL*control and -TFKO cells.
0033 FIGS. 17A - 17J show secretome analysis of AE, AL, 981 , and 974 cells.
0034 FIG. 18 shows the results of an BAB study of AE-control and -TFKO.
0035 FIGS. 19A- 19B show cytokine characterization of APPL-derived exosomes.
0036 FIG. 20 shows off-target analysis of TFKO in APPL cells by RNA-seq.
3.1 Definitions
0037 As used herein, the term ”SH2" refers to an antibody that binds an epitope on the marker CD 105. Thus, cells that are referred tons SH2+ are CD105+. [0020] As used herein, the terms "SH3" and SH4” refer to antibodies that bind epitopes present on the marker CD73, Thus, cells that are referred to as SH3+ and/or SH4+ are CD73+.
0038 As used herein, "immimomodulation" and "immunomodulatory" mean causing, or having the capacity to cause, a detectable change in an immune response, and the ability to cause a detectable change m an immune response;
0039 As used herein, "immunosuppression" and "immunosuppressive" mean causing, or having the capacity to cause, a detectable redaction m an immune response, and the ability to cause a detectable suppression of an immune response.
0040 As used herein, the term "about," when referring to a stated numeric value, indicates a value within plus or minus 10% of the stated numeric value.
As used herein, the term "derived" means isolated from or otherwise purified,
:
For example* placental derived adherent cells are isolated from placenta, The term "derived” encompasses cells that are cultured from cells isolated directly from a tissue, eg., the placenta, and cells cultured or expanded from primary isolates.
0042 As used hereto, toe term “isolated cell,” eg., “isolated placental cell,” “isolated placenta! stem cell,” and the like, itieans a cell that is substantially separated from other, different cells of toe tissue, eg, placenta, front which the stem cell is derived. A cell is “isolated” if at least 50%, 60%, 70%, 80%, 90%, 95%, or at least 99% of toe cells, eg., non-stem cells, with which toe stem cell is naturally associated, or stem cells displaying a di fferent marker profile, ate removed from the stem bell. eg,, during collection and/or Culture of the stem cell.
0043 As used herein, the term " population of isolated cells” means a population of cells that is substantiaily separated from other cells of a tissue, eg., placenta, from which the population of cells is derived.
0044 As used herein, the term “placental cell” refers to a stem cett dr progenitor cell that is isolated from a mammalian placenta, eg. , as described in Section 4, 1 , below, or cultured from cells isolated from a mammalian placenta, either as a primary isolate or a cultured cell, regardless of the number of passages after a primary culture, to certain embodiments, the term “placental cells, “ as osed herein does not, however, refer to frophoblasts, cytotrophoblasts, synchiottdphoblasts, angioblasts, hemangioblasts, embryonic germ cells, embryonic stem cells, cells obtained from an inner cell mass of a blastocyst, or cells obtained from a gonadal ridge of a late embryo, an embryonic germ cell,
0045 As Used herein, a placental cell is “positive” for a: particular marker wheat that marker is detectable above background Detection of a particular marker can, for example, be accomplished either by use of antibodies, or by oligonucleotide probes or printers based On the sequence of toe gene ormRNA encoding toe marker. For example, a placental cell is positive for, eg. , CD73 because CD73 is detectable on placental cells in an amount deiectebly greater than background (in comparison to, e.g., an isotype control). A cell is also positive for a marker when that mark» can be used to distinguish the cell from at least one other cell type, or can be used to select or isolate toe cell when present or expressed by the cell, in the context of, eg., antibody-mediated detection, “positive,” as an indication a particular cell surface marker is present, means that the marker is detectable using an antibody, e.g., a fluorescently-labeled antibody, Specific for thatmarker; “positive” also refers to a cell exhibiting toe matker m an amount that produces a Signal, e.g. , in a cytometer, that is delectably above background. For example, a cell is “CD200* where the cell is delectably labeled with aft antibody specific to CD200, and the signal from the antibody is ttetectably higher than that of a control (e g., badtgrotittd or an isotype control). Conversely, "negative** in the samo context means that the cell surface marker is hot detectable «sing an antibody specific for that marker compared a control (eg., background or an isotype control). Few example, a cell Is “CD34 ” where the cell is not reproducib!y delectably labeled with an antibody specific to CD34 to a greater degree than a control background or an isotype control). Markers not detected. Or not delectable, using antibodies are determined to be positive or negative in a similar manner, using an appropriate control For example, a cell or population Of celis canbe determined to be OCT-4' i f the amount of OCT-4 RNA detected in RNA from the cell or palliation of cells is delectably greater than background as determined, e.g., by a method of detecting RNA such as RT-PCR, slot blots, etc. Unless otherwise noted herein, cluster of differentiation (“CD”) markers are detected using antibodies. In certain embodiments, OCT-4 is determined to be present, and a cell is "OCT-4" if OCT-4 is detectable using RT-PCR.
0046 As used herein, the terms “subject,” “patient,” and “individual” may be used interchangeably to refer to a mammal being treated with a method Of treatment described herein, In a specific embodiment die subject to be treated b a human.
4. DETAILED DESCRIPTION
0047 Provided herein are methods of treating diabetic peripheral neuropathy (DPN) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of tissue culture plastic-adherent placental cells, e.g., placental stem cells, e.g., CD34 , CD 10' , CD 105' , CD200' placental stem cells. In a specific embodiment, said placental cells are formulated as a pharmaceutical composition.
0048 in a specific embodiment, a subject with DPN treated m accordance With the methods provided herein has type I diabetes. In another specific embodiment, a subject with DPN treated in accordance with the methods provided herein has type II diabetes. In certain embodiments, a subject treated m accordance with the methods provided herein has DPN in one or more of die arms, hands, legs, or feet . In certain embodiments, a subject treated in accordance with the methods provided herein has DPN in each of the antis, hands, legs, and feet.
0049 In certain embodiments, a subject treated in accordance with the methods provided herein has DPN and also has a condition that causes a disruption in the flow of blood in the subject’s eripheral vasculature, in a specific embodiment, die sobject hasperipheral arterial disease (PAD). In specific embodiment, the subject bas peripheral vascular disease. lit cermin embodiments, the methods provided hereto result to a detectable improvement of one or more symptoms of DPH to a subject treated to accordance with die methods provided herein. Exemplary symptoms ofDPN include, without limitation, numbness or reduced ability to feel pain or temperature changes, a tingling or burning sensation: to the limbs, sharp pains or cramps, increased sensitivity to touch, muscle weakness, loss of reflexes (e.g., to the ankle), lost of balance and/or coordination, foot problems (such as ulcers, infections, deformities, and bone and joint pain),
:
0051 to certain embodiments, the methods provided hereto comprise admtoistering placental stem cells (e.g., a pharmaceutical composition comprising placental stein ceils) to a subject having DPM in an amount and for a time sufficient for detectable improvement to one or more indicia of impro vement. In a specific embodiment, said indicia of improvement is a change in the epidermal nerve fiber density following treatment, as compared to haseline. Epidermal nerve fiber density is a measurement used to assess toe extent of peripheral diabetic neuropathy* To assess epidermal nerve fiber density, the number of nerve fibers in a skin biopsy is determined. An increase to toe number/density of nerve fibers is indicative of improving neuropathy.
0052 to certain embodiments, toe methods provided herein comprise administering placental stem cells (e.g., a ph&mtaceutical composition comprising placental stem cells) to a subject havtog DPN in an amount and for a time sufficient for detectable improvement to totality of life of the subject asassessed by, e.g., (i)a364tem Short Form Itoalth Survey (SF-36) (see, e.g., Waro et al., Medical Care 30(6):473483); (ii) the Diabetic peripheral neuropathy Scale Shott Fonn (DFS-SF), which measures toe impact of diabetic peripheral neuropathy tot quality of life (see, e.g.. Ban» et al., Phannacoeconomxcs, 2003, 21(17): 1277-90); (in) the Patient Global Impression of Change Scale, to assess changes to neuropathy over time (see, e.g., Kamper et al., 1. Man. Manip. Ther.,2009, 17(3):163-170); and/or <iv) the EuroQol5D (EO-5D™) Scale, which is a health questionnaire used to obtain a descriptive profile and single index value for health status of a patient
0053 In a specific embodiment of toe methods of treatment of DPN described herein, toe placental cells (e.g., a pharmaceutical composition comprising placental stent ceils) are administered by injection. In another specific embodiment of the methods of treatment of DPN described herein, the placental cells (e.g., a pharmaceutical composition comprising placental stem cells) am administered to a subject being treated by imp lantation m said subject of a matrix carscaffold comprising placental cells.
0054 In a specific embodiment of the methods of treatment of DPN described herein, the ptacental cells (e.g, a pharmaceutical composition comprising placental stem cells) are administered intramuscularly . In another specific embodiment, the placental cells (e.g., a pharmaceutical composition comprising placental stem cells) are administered intramuscularly in the area of the DPN (e.g, in one or more of die legs; feet, arms, or hands), In another specific embodiment, the placental cells (e g., a pharmaceutical composition comprising placental stem cells) are administered intramuscularly adjacent to the area of the DPN. In another specific embodiment, the placental ceHs te.g., a pharmaceutical composition comprising placental stem cells) are administered below the knee and above me ankle of a subject that has been diagnosed with DPN. In another specific embodiment Of the methods of treatment of DPN described herein, the placental celts (e.g., a pharmaceutical composition comprising placental stem cells) are administered intravenously. In another specific embodiment of the methods of treatment of DPN described herein, the placental cells (e.g., a pharmaceutical composition comprising placental stem cells) are administered subcutaneously. In another specific embodiment Of the methods of treatment of DPN described herein, the placental cells (e g., aphannaceutical composition comprising placental stem cells) are administered locally. In another specific embodiment of die methods of treatment of DPN described herein, die placental cells (e.g., a pharmaceutical composition comprising placental stem cells) are administered systemicaliy. 0055 In certain embodiments, the methods of treatment of DPN described herein comprise adtmnistration of about 1 x 103, 3 x 103, 5 x 103, 1 x 104, 3 x 104 5 x 104, 1 x 105, 3 x 105, 5 x 105, 1 x 106, 3 x M 5 x W, 1 χ tO7, 3 k 10T, $ x 107 1 x 10*, 3 x lO*, 5 x 10* 1 x 10\5 X Id*, or 1 x 10toplacental cells (eg., as part of a pharmaceutical composition comprising placental stem cells), to certain embodiments, the methods of treatment of DPN described hereto comprise administration of about 1 x 10* to3x Id5, 3 x Id3 to 5 x Id5, 5 x 10s to 1 x Id4, 1 X id4 to 3 x 104, 3 x Id4 to $ x Id4, 5 x Id3 to I x 10s, i x 10s to 3 x id5, 3 x iO$to 5x Id3, 5 x Id5 to 1 X 10*. 1 x I0*to3 x Id6, 3 x lO^to 5 x l<* 5 X Id6 to l X id7, 1 x 107to 3 x Id7.3 x Id7 to 5 x Id1, 5 x ID7 to 1 X ta\ 1 x Id8 to 3 x Id3, 3 x 10* to 5x Id4, 5 x 10* to 1 x id*, 1 x id* to 5 x 10s, or 5 x to 1 x placental cells (eg, as pm of a pharmaceutical composition comprising placental stem cells). In a specific embodiment, the methods of treatment of DPN described herein comprise administration of about 3 x 106 placental cells. In another specific embodiment the methods of treatment of DΡΝ described herein comprise administration of about 1 x 107 placental pells. In another specific embodiment, the methods of treatment of DPN described herein comprise administration of about 3 x 107 placental cells
4.1 ISOLATED PLACENTAL CELLS AND ISOLATED PLACENTAL
CELL, POPULATIONS
0056 The isolated placental cells, sometimes referred to herein as PDACs useful in the methods of treatment of DPN provided herein are obtainable from a placenta or part thereof adhere to a tissue culture substrate and have characteristics of multipoint cells or stem cells, but are not trophoblasts, in certain embodiments, the isolated placental cells useful m the methods disclosed herein have the capacity to differentiate into non-placental cell types.
0057 The isolated placental ceils useful in the methods disclosed herein can be either fetal or matemal tn origin (that is, can have tire genotype of either the fetus or mother, respectively). Preferably, Be isolated placental cells and populations of isolated placental cells ate fetal in origin. As used herein, the phrase “fetal origin” or “hon-matemal in origin* indicates that Be isolated placenta cells or populations of isolated placental cells are obtained from Be umbilical cor dor placental structures associated witit the ferns, l m, that have rite fetal genotype. As used herein, the phrase “maternal in origin” indicates that the cells or populations of cells are obtained from a placental structures associated with the mother , eg, which have the maternal genotype. Isolated placental cells, eg, PDACs, or populations of cells comprising the isolated placental cells, can comprise isolated placental cells Bat are solely fetal dr maternal in origin, or dan comprise a mixed population of isolated placental cells of both fetal and maternal origin; The isolated placental celK and populations of cells comprising the isolated placental cells, can be identified and selected by Be morphological, marker, and culture characteristics discussed below, hi certain embodiments, any of Be placental cells, eg, placental stem cells or placental multipotent cells described herein, are autologous to 8 recipient, eg., an individual who has a DPN. In certain other embodiments, any of the placental cells, eg., placental stem cells or placental multipotent celts described herein, am heterologous to a recipient, eg., an individual who has a DPN. 4.1.1 0 058 The isolated placental cells described herein (PDACs), when culturedin primary caUures or in cCti culture, adhere to the tissue ail tore substrate, &g., tissue culture container surface (eg.; tissue culture plastic), or to a tissue cufctoe surface coated with extracellular matrix or ligands such as laminin, collagen (eg. , nativeor denatured), gelatin, fibronectin, Ornithine; vitronectin, and extracellular membrane protein (e.g., MATR!GEL4KBD Discovery l4bware» Bedford, Mass,)). The isolated placental cells in culture assume a generally fibroblastoid, stellate appearance, with a number of cytoplasmie processes extending from the central cell body. The cells are, however, morphologically distinguishable from fibroblasts cultured under the same conditions, as the isolated placental cells exhibit a greater number of such processes than do fibroblasts. Morphologically, isolated placental cells are also distinguishable from hematopoietic stent cells, Which generally assume a more rounded, or cobblestone, morphOTOgy in culture.
0059 In certain embodimen ts, the isolated placental cel ts useful m the methods disclosed herein, when cultured in a growth medium, develop embryoid-tike bodies. Embryotd- like bodies ate noncontiguous dumps of polls that can grow on top of ail adherent layer of proliferating isolated placental cells» The term “embryoid-like” is used because the clumps of cells resemble embryoid bodies, clumps of cells that grow from cultures of embryonic stem cells Growth medium in which embtyoid-ltke bodies can develop ίn a proliferating culture of isolated placentalcells includes medium comprising, c.g., DMEM-LG (e.g., from Gibco); 2% fetal calf eenira (e.g.. from Hyclone Labs.); tx raHilm-tramfemn-sckmum (ITS); lx linoleic acid-bovine serani albumin (LA-8SA); 10-9 M dexamethasone ( e.g.;from Sigma); 10-4 M ascorbic acid 2- phosphate ie.g., from Sigma); epidermal growth factor 10 ng/mL (e.g., from R&D Systems); and platelet-derived growth fector(PDGF-BB) 10 ng/mL ( e.g., from R&0 Systems).
4.1.2 Cell Surface. Molecular and Genetic Markers
0060 The isolated placental Cells, isolated raultipotehtplaeental cells or isolated placental stem cells, and populations of such isolated placental cells, useful in the methods disclosed herein, e.g., the methods of treatment of a DPN of a subject, are tissue culture plasiic- adherent human placental cells that have characteristics of mulnpotent cells or stem cells, and express a plurality of markers that can be used to identify and/or isolate the cells, or populations of cells feat comprise the stem cells. In certain embodiments, fee PDACs are angiogenic, in vitro or in vivo. The isolated placental cells, and placental cell populations described herein (that is, two or more isolated placental cells) include placental cells and placental cell-containing cell populations obtained directly from the placenta, or any part thereof (e.g,, chorion, placental cotyledons, or the l ike)- Isolated placental cell populations also include populations of (that is, two or mom) isolated placental cells in culture, and a population m a container, e.g., a bag. The isolated placental Cells described herein am not bone narrow-derived mesenchymal cells, adipose-derived mesenchymal stem cells, or mesenehymal cells obtained from umbilical cord blood, placental blood, of peripheral blood: Placental Cells, e.g., placental multipotent cells and placental cells, useful in the methods and compositions described herein are described herein and, e.g., m US. latent Nos, 7,311,904; 7,311,905 and 7,468,276: and in US; Patent Application Publication No. 2007/0275362, the disclosures of which are hereby incorporated by reference in their entireties.
0061 In certain embodiments, the isolated placental cells are isolated placental stem cells, In certain Other embodiments, the isolated placental cells are isolated placental multipotent cells. In one embodiment, the isolated placental cells, e g, PDACs, are CD34', GD 10* and CD 105' as detected by tiow cytometry. In another specific embodiment, the isolated CD34 ;
£D10% CI>I05* placental cells have the potential to differentiate irito cells of a neural phenotype, ceils of an osteogenic phenotype, and/or cells of a chondrogenic phenotype. In another specific embodiment, the isolated CD34' CD10+ CD105 ; placental cells ate additionally CD200’. In another specific embodiment, the isolated CD34 , CD 10% CD 105 ’ placental cells ate additionally CD45 or CD90+. In another specific embodiment, die isolated CD34% CD10% CD 105* placental cells are additionally CD45" and CD90' as detected by Sow cytometry- In another specific embodiment, the isolated CD34 , CD 10+ CD 105", CD20T placental cells are additionally CD90* or CD45% as detected by flow cytometry. In another specific embodiment, the isolated CD34' CD 10 % CD105', CD200+ placental cells are additionally CD90* and CD45 ; as detected by flow cytometry, i.e., the cells ate CD34' CD10' CD45% CD90' CD105' and CD200' In another specific embodiment, said COM' , CD10% CD45 , CD90', CD105 · , CD200' cells am additionally CD80 and CD86 ,
160621 In certain embodiments, said placental cells are CD34' CD10 , GD105' and CD200' and one or more of CD38' CD45 , GD80 , CD86 , GDI 33' HLA-DR,DP,DQ , S$BA3% SSEA4' CD29% CD44% CD73% CD90% CD 105", HLA-A,B,C% PUU% ABC-p% and/or OCT-4*, as detected by flow cytometiy, to other embodiments, any of the CD34 ,
CD 10* CD 105' cells described above are additionally one or more of CD29 VCD38 , CD44', CD54' , SH3+ or SH4'- to another specific embodiment, the cells are additionally CD44+, to anotherspeeiftc embodiment of any of the isolated CD34 , CD10*. CD105+ placental cells above* the cells are additionally one or more of CD117, CD133", KDR (VEGFR2 ), HLA- A,B,C+ HLA-DP*DQ,DR Programmed Death- 1 Ligand (PDLl)+, or any combination thereof
0063 In another embodiment, the CD34~, CD104* CDlOS* cel¼ ate additionally one or more of CD13n CD29+, CD33+CD38-, C D44+ CD45-, CD54+, CD62E-, CD62L-,
CD62P-, SH3+ (CD73-+) SH44 (CD73+), CD80-,CD86-, CD90+, SH2+ (CD105 +),
CD 106/VCAM+ CD117-, CD 144/VE-cadherinlow, CD184/CXCR4-, CD200+, CD133-, OCT- 4+, SSEA3-, SSEA4-, ABC-p+, KDR-(VEGFR2-) HLA-A,B,C+, HLA-DP,DQ,DR- HLA- G-, or Programmed Death- 1 Ligand (PDL 1 )+, or any combination thereof, to a other embodiment, the CD34-, CD10+, CD105+ cells am additionally CD13 +, CD29+, CD33+ CD38-, CD44*, CD45-, CD54/ICAM-H CD62E-, CD62L-, CD62P~, SH3+ (CD73+), SH44 (CD734), CD80-, CD86-, CD90+, SH2+ (CD 105-+ ), CD106/VCAM+, CDl 17-, CD144/VE- cadherinlow, CP184/CXCR4-, CD200+, CD133-, OCT-43·, SSEA3-, SSEA4-,ABC-p+,
KDR~ (VEGFR2-), HLA-ABC+, HLA-DP,DQ,DR- HLA-G-,and Programmed Death-1 Ligand (PDM)t.
0064 Inanother specific embodiment, any pf the placental cells described herein are additionaHy ABC-p4, as detected by flow cytometry* or OCT-44 (POU5FB), as determined by RT-ECR, wherein ABC-p is a placenta-specific ABC transporter protein (also known as breast cancer resistance protein (BCRP) and as mitoxamrone resistance protein (MXR)), and QCT-4 ½ toe Octaroer-4 protein (PO05P1), In another specific embodiment, any pf toe placental cells described herein are additionally SSEA3- or SSEA4-, as determined by flow cytometry, wherein SSEA3 is Stage Specific Embryonic Antigen 3, and S$EA4 is Stage Specific Embryonic Antigen 4. In another specific embodiment, any of the placental cells described harem are additionally SSEA3- and SSEA4-.
0065 to another specific embodiment, any of die placental cells described heroin are additionally one or more ofMHC-B (e.g.* HL A-A,B,C4), MHC-II- (e.g.HLA -DP,DQ,DR-) or HLA-G-. in another specific embodiment, any of the placental cells described herein are additionatly one or more of MHC-l t (e.g., BLA-A,B,C*), MHC-lf-(> g,, HLA-DP,DQtDR-) and HlArG-.
0066 Also provided herein are populations of the isolated placental cells, or populations of cells, e.g., populations of placental cells, comprising, e.g., that ate enriched for, the isolated placental ceils, that are useful ih the methods and compositions disclosed herein. Preferred peculations of cells comprising the isolated placental celts, whereih the populations of celts comprise, e.g,, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 6<¾ 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% isolated GD104, GD105-4 and CD34- placental cells; that is, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% 70%, 75%, 80%, 85%, 90%, 95% or 98% of cells m saidpopuiation are isolated CDJOr, CD105* and CD34- placental cells. In a specific embodiment, the isolated CD34--, CD10+-, GplOS^ placental cells areadditionally CD200+; In another spectfic embodiment the isolated CD34~ ; CD 10*, CDI05*, CD20O* placental cells are additionally CD90+ or CD45- , as detected by flow cytometry, hi another specific embodiment, the isolated CD34-, CD 10*. CD 105*, CD20CH- placental cells are additionally CD90* and CD45-, as detected by flow cytometry. In another specific embodiment, any Of the isolated CD34-, GDI 0*, CD105+ placental cells described above are additionally one or more ofCD29*, CD38-, CD44*, CD54+, $H3+ or SH4 k In another specific embodiment, the isolated CD34-. CD10*, CD 105* placental cells, or isolated CD34-, CDi(H, GDI 05*, CD200* placental cells, areadditionally C¾M44, In aspecific embodiment Of any of the populations of cells comprising isolated CD34-, CD 10* GDI 05- ptacental cells above, the isolated placental cells ate additionally one or mote Of CD13*, CD29*, CD334, CD38-, CD44* CD45-, CDS4*, CD62E-, CD62L-, CD62P-, SH3* (CD73*), SH4t (CD73*), CD80-, CD86-, CD90*, SH2* (CD105+), GDI 06/VCAM4, CD 117- GDI 44/VE- cadherinlow, CD184/CXCR4-, CD200H CD133-, OCT-4+, SSEA3™, SSEA4™, ABC*p+,
KD&*- <VEGFR2~), ΗΕΑ-Α,Β,Ο*, HLA>DP,DQ,DR-r HLA-G-, or Programmed Death-1 Ligand (PbLtK ot any combination thereof. In another specific embodiment, the CD34",
CD to*, GDI 05* cells are additionally CD13*, CD29+, CD33+, CD38-, CD44*, CD45- CD54/ICAM*, CD62E-, CD62L-, CD62P-, SH3* (CD73*), $H4*(CD73*), CD8O~vCD86~, CD90*, SH2*(CD105*K CD106/VCAM*, CDU7 > CDi44/VE~cadheriolow?
CmW€Xm*~, CD2Q0*. CD133-, OCT-4¾SSEA3-, SSEA4-, ABC^ KDR™ (VEGFR2-), MtA-AXC'^ HLA-DP,DQ,DR-> f$lA>G-v»»d PmgmmmedDeath-l Ligand CPDUJ4 0067 to certain embodiments, the isolated placental cells useful in the methods and
Compositions described herein are Ohe of more. Or all. Of CD 1.04, CD294, C¾34-> CP38- , CD444 CD45-, GD54+, CD90+, SH24 SH34, SH4+, SSEA3-, SSEA4~# OCT4+, and ABC- p¾ wherein said isolated placental cells are obtained by physical and/of bnzymatic disruption of placental tissue. In a specific embodiment, the isolated placental cells are OCT-44 and ABC-p4 M another specific embodiment, the isolated placental cells am OCT-44 and CD34-, wherein said isolated placental cells have at least one of the Mowing characteristics: CD 104, CD29+* CD444, CD4S-, CD54t, CD904, $H34 $M44, $S£A3~> and SSEA4-. to another specific embodiment, the isolated placental cells are OCT-44, CD34-, CD 104, CD29+, CD444, Cp45~, CD544;, GD9CI4, SH3+, SH44, SSEA3-, add SSEA4-. to another embodiment, toe isolated placental cells are OCT-44, CD34-", SSEA3--, and SSEA4™. to another specific embodiment, the isolated placental celts are OCT-44 and CD34-, and ½ either SH24 bf SH34. in another specific embodiment, the isolated placental tolls are OCT-44, CD34-, SH24, and SH34. to another specific embodiment, the isolated placental ceils are OCT-44, CD34-, SSEA3™, and SSEA4-, and are either SH2+ or SH3-t . In another specific embodiment, the isolated placental cells are OCT-44 and CD34-, and either SH24 or SH34, and is at least one of CDfOt, CD294. CD444, CD4S-, CD544, CD904, SSEA3-, or SSBA4-. In another specific embodiment, die isolated placental cells are OCT-44, CD34-, CD1CH-, CD294, CD44+ CD45~, CD544, CD904,
SSEA3-, and SSEA4-, and either SH24 or SH34.
0068 in another embodiment, the isolated placental ceils useful in the methods and compositions disclosed herein are S824, SH34, SH44 and OCT-44, In another specific embodiment· the isolated placental cells are CDiO*, CD20+* CD44+, CDS44 CD90+, CD34- CD45-, S^EA3~, or SSEA4-:, to another embodiment, the isolated placerital cells are SB24, SH34, SH44, SSEA3-T said SSEA4-. In another specific embodiment, the isolated placental cells are Sti24, SH34, SH4+, SSEA3- and SSEA4— , CD104, CD294, CD444- CD54+ CD904, OCT-44, CD34™ orCD45~.
0069 in another embodiment, the isolated placental cells useful in the methods and compositions disclosed herein areCDlO*, CD294 CD34-, CP444> CD45-, CD544 CD904 7H2-L SH3*, and SH4+; wherein said isolated placental cells are additionally one or more Of GCT-4+/SSEA3- or SSEA4-.
In certain embodiments, isolatedplacental cells usefiil to the methods and
Compositions disclosed herein are CD200* or HLA*G~. In ¼ ^pecMc embodunent, the isolated placental cells ate CQ200+ add HLA-CK In another specific embodiment, the isolated placental cells are additionally CD73* and CD1054, In another specific embodiment, the isolated placental pells are additionally CD34-, CD38- or CD45-. In another specific embodiment, the isolated placental cells are additionally CD34-> C 038~ aod C D45~. in another specific embodiment, said stem cells are CD34-, CD38-, CD45-, CD73+ and CD1O5+. In another specific embodiment, said isolated CD20O+ or liLA-G- placental cells facilitate the formation of embryoid4ike bodies ina population of placental cells comprising die isolated placental cells, under conditions that allow the formation of embryoid-like bodies. In another specific embodiment, the isolated placental cells are isolated away from placental ceils that are not stem orinitltipotent cells, in another specific embodiment, said isolated placental cells are isolated away from placental cells that do not display these markers. 0 0 71 another embodiment, a cell population useful in die methods and compositions described jbe*¾m i$ a population of cells comprising, e.g., that is enriched for, CD200+, HLA-G- stem celts. to a specific embodiment, said population is a population of placental cells; In various embodiments, at least about 10%, at least about 20%, at least abont 30%, at least about 40%, at leas* about 50%, or at least about 60% of cells m said cell population are isolated CD200 K HLA-G- placental cells. Preferably, at least about 70% of cells in said cel l population are isolated CD200+, HLA-G- placental cells. Mine preferably, at least about 90%, 95%, or 99% of said cells are isolated CD200+* HLA-G- placental Cells, to a specific embodiment of the cell populations, said isolated CD20O+, HLA-G- placental cells are also CD73+ and CDI05+.
In another specific embodiment, said isolated CD200+, HLA-G- placental cells are also GD34-, CD3S- or CD45-, to another specific embodiment, said isolated CD200*, HLA-G- placental cells a* also CD34-, CD38-, CD45-. CD73+ and CD 105+. In another embodiment, said cell population produces one or more embryoid-like bodies when cultured under conditions that allow the formation of embryouMfire bodies. In another specific embodiment, said cell population ¼ isolated away from placental cells that are not stem cells, to another specific embocfimeni. said Isolated CDiOOk HLA-G- placental cells are isolated siway from placental cells that db not display these markers.
0072 In another embodiment, the isolated placental cells tisefid to tire methods and
Compositions described: hereto are CD73k CD1 OSk and CD200k to another specific embodiment, toe isolated placental cells ate HLA-0-. toanother specific embodiment, toe isolated placental cells are CDM^ CD38- or CD4$~> to another specific embodiment, toe isolated placental cells ate CD34-, CD3fi-abd CD45- toanother specific embodiment, toe isolated placenta) cells are CD34-, €D3$-« CD45-. and HIA-G~. to anodier specific emboditnent, toe isolated CD734; CD105k and CD2004 placental cells facilitate toe formation of one or more embryoid-like bothes in a population of placental cells comprising toe isolated placental cells, when toe population is cultured under conditions that allow the formation of embryoid-like bodies, to another specific embodiment, the isolated placental cells are isolated axyay from placental cells that are not toe isolated placental cells, to another specific embodiment, the isolated placental cells are isolated away from placental Cells that do not display these markers;
0073 In another embodiment, a cell population useful to toe methods and compositions described herein i$ a popuWon of cdls comprising, e.g., that is enriched for, isolated CD73+, CD105k CD200+ placental cells. In various embodiments, at least about 10%, at least about 20%, at least about 30%; at least about 40%, at least about 50%, or at least about 60% of cells to said cell population are isolated CD73t, CD 105k CD20Pt placental cetis. In another embodiment, at least about 70% Of said cells in said population of cells are isolated CD734,
CD 105k 00200* placental cells, to another embodiment, at least about 90%, 95% or 99% of cells in said population of cells are isolated CD73*, CD 105k CD200* placental cells, to a specific embodiment of said populations, die isolated placental cells are HtA-Ck toanother specific embodiment, toe isolated placental cells me additionally CD34-, CD38- or CD45-. to another specific embodiment, the isolated placental cells are additionally CD34-, CD38- and CD45-. to another specific embodiment, toe isolated placentid cells are additionally CD34~y CD38k, CD45-', and HLA-G-.. to another specific embodiment, said population of cells produces one or more embryoid-like bodies when cultured under conditions that allow toe formationof embryoid-like bodies, to another specific embodiment, said population of placental cells is isolated away from placental cells that are not stem cells, to another specific embodiment,said population of placental ceila is isolated away fitow placental cells that do not display these characteristics.
0074 in certain other embodiments, the isolated placental cells are one or mote of
GP!Ot, CD29r, CD34-, CD38~, CD44+, CD4S-* CD54+>CD9tH, SH2+1 SH34, SH44, SSBA3-, SSBA4-, OCT44, HLA-G- or ABC-p* in a specific embodiment, the isolated placental cells are CDlti-f, CD29+, CD34-, CD38-, CD444, C P 45 · , CD544, CD904, SH24;, SH34, SH4t, SSE A3-, SSEA4-, and OCT-4*. In another specific embodiment, the isolated placental cellsare CDl^tCD294, C?D34-, CD38-. CD45-, CD54* SB24, and SH4*. in another specific embodiment, the isolated placental eelfe are CD10*, CD294, CP34r-, CD3#~, CD4>~, CD544, $B24 , Sip*, SH44 and QCT-44. In another specific embodiment, die isolated placental cells are CDI04, CD29+, CD34™, CD38-, CD44+, CD45- CD544-, CD904, HLA-G-, SH24, SH3t, SH44. In another specific embodiment, the isolated placental cells are OGT-4-f and ABOp-h In another specific embodiment, the isolated placental cells are SH24„ SB34, SH44 and OCT44. to another embodiment, the isolated placental cells are OCT-44% CD34-, SSKA3 -, and SSEA4-. to a specific embodiment, said isolated OCT-44·, CD34-, SSEA3-, and SSEA4" placental cells are additionally CTU04, CD29+, CD34-, CD444, CD45-, CD544, CD90+ SH24 , SH34, and SH4*. to another embodiment, the isolated placental cells ate OCT- 44· andCD34-, and either SH34 or SH44-. to another embodiment, the isolated placental cells ate €034- and either CD1 tit , CD294, CD444 , CD544, CD90* or OCT-44.
(00751 Inanother embodiment, the isolated placental ceils useful in the methods and compositions described berm are CD2QCH- and OCT-44 , to a specific embodiment, the isolated placenta! Cells are CD734 and €01054; to another specific embodiment, said isolated placental cells are HLA-G-. to another specific embodiment, said isolated GD200* 0CT44 placental cells are CD34· ·, CD38« or CD45-. to another specific embodiment, said isolated 002004-, QCT4+ placental cells ate CD34-, CD38- and CD45-. to another specific embodiment, said isolated CD200* OCT44 placental cells are CD34-, CD38-, CD45- CD73*, CD 105* and HLA*G~. tn another specific embodiment, the isolated CD200+, 0CT4-4 placental cells ffccilflate die production of one or more embryoid-like bodies by a population of placental cells that comptises the isolated cells, when the population is cultured muter conditions that allow the formation of embryoid-Uke bodies, to another Specific embodiment, said isolated CD200*» OCT44 placental cells are isolated away from placental cells that are not stem cells, to another spedfic embodiment, said isolated CD2004, OCT-4+ placental cells are isolated away trom placental cells that do not display these characteristics.
0076 In another embodiment, a cell population useful in toe methods and compositions described hetoto is a population of cells comprising, e.g„ that is entiched for, 032004, QCT-44 placenta! cells, to various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60% of cells, in said cell population are isolated GD200*, OCT-44 placental cells, to another embodiment, at least about 70% of said celts am said isolated CD2QG4, GCT-4+ placental cells;, to another embodiment, at least about 80%, 90%, 95%, or 99% of cells to said cell population are said isolated CD2O0% OCT-44 placentai cells. to a specific embodiment of toe isolated populations, said isolated CD2004> OCT-44 placental cells are additionally CD734 and CD 1054-. to another specific embodiment, said isolated CD2904, 0CT-44 placentai cells are additionally HLA-G-. to another specific embodiment, said isolated CD2004, OCT-44 placental cells are additionally CD34-, CD38- mid CD45-. to another specific embodiment, said isolated CD2004, QCP44 placental cells are additionally CD3A-, CD38- , CD45-, CD734, CD 1054 mid HLA-G-. to another specific embodiment, the cell population produces one or more embryoid-like bodies when culttoed under conditions that allow toe formation of erohryoid-tike bodies. In another specific embodiment, said cell population is isolated away from placental cells that are not isolated CD2004 ; dCT-44 placental cells, to another specific embodiment, said cell population is isolated away from placental cells that do apt display these markers.
0077 In another embodiment, the isolated placenta! cells usefiil to the methods and compositions described herein are CD734, CD1054 and HLA-G”. to another specific embodiment, the isolated CD73t, CDI054 and HLA-G- placental cells are additionally CD34-, GD38-- or CD45 to anodier specific embodiment, the isolated CD?3+v GD 1054-, HLA-G- placental cells are additionally CD04-, CJD38- and CD45-. to another specific embodiment, the isolated CD73+* CDi05+, HLA-G-placental cells are additionally OCT-44 , to another specific embodiment, the isolated C)E>734, CDlOS+v j¾LA-(3~ placental cells are additionally CD2004. to smother specific embodiment, the isolated CD734, CD105+, MLA-G- placental cells are additionally CD34-, CD3S- , CTM5~,OCT*44 and CD2004. to another specific embodiment, die isolated CD73+, CD! 054, HLA-G- placental cells facilitate the formation of embryoid-like bodies in a population of placental cells comprising said cells, when die population is cultured under c<todition$ that aittiw the formation of embryoid-like bodies. In another specific embodiment, said the isolated CD73+, CD105+, HLA-G*- placental cells are isolated away from placental celts that are not the isolated CD73+, CD 1054 , HI A-G™ placental cells. In another specific embodiment, said the isolated CD73+, CD105+, HLA-G- ptacental cells are isolated away from placental cells that dp not display these markers.
0078 to another embodiment,* ceUpoptoation usefiit id (he methods and compositions described herein is a population of cells comprising, e.g>, that is enriched for, isolated CD73+, CDlOSt and HLA*G~ placental cells. lh various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least: about 60% Of cells ip said population of cells are isolated CD73+, CD 185% HLA-G~ placental celts, to another embodimenr, at least about 70% of cells in said population of ceils are isolated CD73+, CD105+, HLAGh placental cells, to another embodiment, at least about 90%, 95% or 99% of celts in said population of cells are isolated CD73+, CDI05+, HLA-G™ placental cells. In a specific embodiment of the above populations, said isolated CD73+, €D105+, HLA-G- placental cells are additionally CD34-, CD3&- or CD45-. to another specific embodiment, said isolated CD73+, CDI05+, HLA-G- placental cells are additionally CD34-, CD3&- and CD45-, to another specific embodiment, said isolated CD73+, CD 105% HLA-G™ placental cells are additionally OCT-4+. In another specific embodiment, said isolated CD73+, CD 1G5+, HLA-G™ placental celts are additionally €D2O0% in another specific embodiment, said isolated CD73+, CD105% HLA-G- placental cells are additionally CD34-, CD3&-, CD45-, OCT-4+ and CD200+. to another specific embodiment, said cell population is isolated away from placental cells dim are not CD73+, CD1G5+, HLA-G- placental cells, to another specific embodiment, said cell population « isolated away from placental cells that do not display these markers.
0079 In another embodiment, the isolated placental cells useful in the methods and compositions described herein are CD73+ and CD1G5+ and facilitate the formation of one or mote embryoid-like bodies in a population of isolated placental cells comprising said CD73+,
CD IG5+ cells when said population ¾ cultured tinder condition that allow formation of embryoid-like bodies. In another specific embodiment, said isolated CD73+, CD 105+ placental cells are additionally CD34-, C3D38-- or CD45- . In another specific embodiment, said isolated CD73+, CD105+ placental cells am additionally CD34-, CD38- and CD45-. In another specific embodiment, said isolated CD73+, CDi 05+ placental cells are additionally OCT-4+. to another specie embodiment,$aid i$olatedCD73H CD 105+ placental pells ate additionally ΟθΜ+, CD34-, CD3&- and CD45™, In another specific embodiment, said isolated CD73+, CD105+ placental cells ate isolated away from placental cells that are not said cells. In another specific embodiment Said isolated CD73*, CD 105+ placental cells are isolated away from placental cells that do not display these characteristics.
In another embodiment, a cell poptilation useful iii the methods and compositions described herein is a population of cells comprising, e.g>, that is enriched for# isolated placental cells that are CD73+; CD105+ and facilitate the formation of one or more embryokMike bodies in a population of isolated placental celts comprising said ceils when said population is cultured under conditions that allow formation of embryoid-like bodies» 10 various embodiments, at least about lb%# at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 00% of cells in Said population of cells ait said isolated CD73fj CD105+ placental cells. In another embodiment# at least about 70% of cells in said population of cells are said isolated CD'73-f, CD 105+ placental Cells. In another embodiment, at least about 90%, 95% or 99% of cells in said population of cells are said isolated CD73+, CDIG5+ placental cells. In a specific embodiment of the above populations, Said isolated CD73+, CD105+ p½cental cells are additionally CD34-, 0>3&- or CD45 v tn another specific embodiment, said isolated CD73+, CD 105+ placental cells are additionally CD34™, CD3B™ and CD4S-. In another specific embodiment, said Isolated CT>73+> CD105+ placental cells ate additionally OCT-44·, In another specific embodiment, said isolated CD73+, CD105+ placental cells are additionally CD200+. In another specific embodiment, said isolated CD73+, CD105+ placental cells are additionally CD34-, CD38K CD45-", OCT-4+ and CD200+. In another specific embodiment, said cell population is isolated away from placental cells that are not said isolated CD73+, CD 105+ placental cells, in another specific embodiment, said cell populatitm is isolated away from placental cells that do not display these markers.
0081 In another embodiment, the isolated placental cells useful in the methods and compositions described herein are OCT-4+ and facilitate formation of one or more embryoid-like bodies in a population of isolated placental cells comprising said ce lls when cultured under conditions that allow formation of embryoid-like bodies. In a specific embodiment, said isolated QCT-4+ placental cells are additionally CD73+ and CD105+. In another specific embodiment, said isolated QCT-4+placental cells are additionally CD34- , CD38-, Or CD4S ·- In another specific embodiment, said isolated 0CT4* placental cells are additionally CD20O*. fi* another specific embodiment, said isolatedOCT-4* placenta! cells are additionally CD73*, CD! 05*, CD20Ot, CD34-, OD30-, and CD45-. In another specific embodiment, said isolated OCT-4-5 placental celts are isolated away from placental celts that are not OCT-4* placental cells- In another specific embodiment, said isolated CfcT-4* placental cells are isolated away fiord placentaleells thai do not display these characteristics.
In another embodiment, a cell population useful in the methods and compositions described herein is a population of cells comprising, e.g., that is enriched for, isolated placental cells that are OCT-4* and facilitate the formation of one or nwe embiyoi^lijke bodies to a population of isolated placental cells comprising said cells when said population ¼ cultured under conditions that allow formation of embryoid-lifce bodies, to various embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least abdot 50%, or at least about 60% of cells iu said population of cells are said isolated OCT-44 placental cells. In another embodiment, at leasf about 70% of cells in said population of cells are said isolated OCT-4* placental cells. In another embodiment, at least about 80%, 90%, 95% or 99% of cells in said population of cells are said isolated OCT-4* placental celts. In a specific embodhrieUt of the above populations, said isolated OCT-4* placental cells are additionally CD34-, CD38- or CD45-. In another specific embodiment, said Isolated OCT-4* placental cells are additionally CD34-, CD38- and CD45-i In another specific embodiment, said isolated OCT-4* placental cells are additionally CD73+ and CD105*. to another specific embodiment, said isolated OCT- 4* placental cells ace additionally CD200*. In another specific embodiment, said isolated OCT- 4* placental cells are additionally CD73*, CD 105*, GD200*, CD34-, CD38~, and CD45- to another specific embodiment, said cell population is isolated away ftoro plaeental celts that are not Said cells, to another specific embodiment; said cell population ½ isolated away from placental cells that do not display these markers.
{90831 In another embodiment, the isolated placental cells usefiti ½ the methods and compositions described herein are isolated HLA*A,B,C* CD45~vCD133~ and CD34~ placental cells, to another embodiment, a cell population useful in the methods aud eompositions described herein is a population of cells comprising isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% of at least about 99% of cells in said isolatedpopnlation of cells are isolated ELA-A,B,C+, GD45 , CD 133- and GD34- placental cells. In a specific emboditoent, said isolated placental cell or population of isolated placental cells to isolated away from placentaicells that are not HLA-A,B,0, CD45-, CD 133- and CD34- placental cells. In another specific embodiment, said isolated placental cells are non* maternal m origin , In another specific embodiment said isolated population of placental cells are substantially free of maternal components; e.g,, at least about 40%, 45%, 5-0%, 55%, 60%*
65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% Or 99% ofsaid cells in said isolated population of placental cells ate non-materoal to origin.
0084 In another embodiment, the isolated placental cell! useful in the methods and compositions describedherein are isolated CDIOf, <Γ013-Κ€033+, CD45-, CDi l7- and CD 133- plaeenral cefls. In another embodiment, acell population useful in the methods and compositions described hereto is a population of ceils comprising isolated placental cells. whereto ar least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells to said population of celts are isolated CDI Or, CD! 3% CD334, CD45-,
CDl 17- and CDI 33- placental cells, to a specific embodiment, said isolated placental cells or population of isolated placental cells to Isolated away from placental cells that are not said isolated placental cells, to another specific emboditoent, said isolated CDltH·, CD13+, CD33+, C045-, CDl 17- and CD133- placental cells ate non-matemal in origin, i.e., have the fetal genotype, In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells to said isolated population Of placental cells, are non-matemal in origin. In another specific embodiment, said isolated placental cells or population of isolated placental cells are isolated away from placental cells that do not display these characteristics,
{β085| to another embodiments the isolated placental cells useful to the methods and compositions described hereto are isolated CDl 0-, CD33- , CD44+, CD45- , and CD 117 - placental cells. In another embodiment, a cell population useful for the hi the methods and compositions described herein is a population of cells comprising, e.g., enriched for, isolated placental cells, wherein at least about 70%, at least about 80%, at least about 90%, at least about 95% or at least about 99% of cells to said population of cells are isolated CD to*-, CD33-, CD44+, CD45 -, and CDl 17- placental cells, to a specific embodiment, said isolated placental cell or population of isolated placental cells is isolated away from placental cells that are not said cells. In another specific embodiment, said isolated placental cells am non-matemal in origin. In anoto specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, &%, 70%, ?5%, 90%, 85%, 90%, 95%, 98% or 99% Of Said cells in said cell population are non-matemal in origin. In another specific embodiment, said isolated placental cell or pqptdation of isolated placental cells is isolated away from placental cells that do not display these markers.
100861 to another embodiment, the isolated placental cells useful in tire methods and compositions described heroin ate isolated CDIOT, CD'13-% C033K CP45-, and CD 117» placental cells, to another embodiment, a cell population useful for in the methods and compositions described herein is a population of cells comprising, e.g,, enriched ¾, isolated CD 10™, CD ! 3-, CD33™, CD45-, and CDU?~ placental ce¾ wherein at least about 70%, at least about 80%, at least abom 90%; at least about 95% or at least about 99% of cells in said population are CD 10-, CDI 3~, CD33-, CD45™, and CDI 17- placental cells. In a specific embodiment, said isolated placental dells Or population of isolated placental cells are isolated away from placental cells that are not said cells. In another specific embodiment, said isolated placental Cells are non-matemal in Origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are non-materaal in Origin. In ahpther specific embodiment, said isolated placental cells or population of isolated placental cells is isolated away from placental cells ¾t do not display these characteristics.
100871 to another embodiment, die isolated placental Cells useful to the methods and compositions described heroin are HLA Α,Β,<¾ €D45~, CD34™, and CD133™, and ate additionally CD 10+, CD13+, CD38*, CD44t, CD9(Hv CD 1054, CD20CH and/or HLA-G-, and/or negative tor GDI 17. In another embodiment, a cell population useful in the methods described herein is a population of cells comprising isolated placental cells, wherein at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 95%, 98% car about 99% of fite cells in said population tune isolated placental cells that are HLA Α,Β,Ο, €D45~, CD34™, CD133™, and that are additionally positive for CD 10, CD13, CD38, CD44, CD90, CDI05, CD200, and/or negative for CDI 17 and/or HLA-G. to a specific embodiment, said isolated placental cells or population of isolated placental cells are isolated Sway ¼ro placental cells that ate not said cells, !n another specific embodiment, said isolated placental cells ate non-matemal in origin, to another specific embodiment, at least about 40%^ 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% Of said cells to said cel! population are non-matemal in origin. In another specific embodiment, said isolated placental cells or population of isolated placental cells are isolated away from placenta! cells that do not display these markets-
160881 In another embodiment the isolated placental cells tisefid in the methods and compositions described herein are isolated placental cells that are CD2QQ4 and CD104, as determined by antibody binding, and GDI 17-, as determined by both antibody binding and RT- PCR. in another embodiment the isolated placental cells useful in the methods and compositions described herein are isolated placental ceils, e,&, placental stent cells or placental muhipotentcells, that are CDKH-, CD29-, GD544, CD2004-, HLA-G-, MHC class 1+ and jh-2- microglobylin^ In another embodiment, isolated placenta! cells usefulin the methods and compositions described herein are placental cells wherein the expression of at least one cellular marker is at least two-fbld higher titan tbra mesenchymal stem celt (e.g., a bone marrow-derived mesenchymal stem cell). In another specific embodiment, said isolated placental cells are non· maternal in origin. In another specific embodiment, at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 90%, 85%, 90%, 95%, 98% or 99% of said cells in said cell population are ήοη-maiemal inorigin.
Inanmher embodiment, the isotered placental cells useful in the methods add compositions described herein are tsolated placental cells, e.g., placental stem cells or placental omnipotent cells, that ate tine or mote ofCDI0% CD29+, CD444, CD45-, CD54/1CAM4, CD62E-, CD62L-, CD62P-, CD8<K CD86-, CD103-, CD104-, GDI 054, CD 1CWVCAM+ CD144/YE-cadherinjow, CD184/CXCR4-, p2^nicroglobolintow, MHC-Ilow, MHC41-, HLA- Glow, and/or PDLllow. In a specific embodiment,, the isolated placental ceils ate at least CD29+ and CD54K in another specific embodiment, the isolated placental cells ate at least CD44+ and CDI064, In another specific embodiment, the isolated placenta} cells ate at least CD29-K
160901 In another embodiment, a cell population useful in the methods and compositions described herein comprises isolated placental cells, and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the cells in said cell population are isolated placental cells that are one or rooreof CD!On CD29+, CD44-f, GD45- , CD54/ICAM-r, CD62-E-, CD62-L-, CD62-P -,
CD80-, CD86-, GDI 63-, CD104-, CD1054, CD106/VCAM^ CD144^E-Cadhetmdim, CDE84/CXCR4'··, p2-microglobulindim, HLA-Mim, HLA-H , HLA-Gdim, and/or MSLldim; In anotoerspecific embodiment, at least 50%, 60%, 70%, 80%, 90%, >5%, 98% or 99% of cells to said cell population are CD10% CP29*, CD44+, CD45-r, CD54/ICAM% CD62-E-V CD62-L-, CD62-P™, CD8G- CD$6-~, CDl 03- CD104- CD10S^, CD 106/VCAM^ CDH4/VE- cadherindim, CD184/CXCR4 , p2-microglobulindim. MHC-ldim, MHC-IL. HLA-Gdim, atid PDLldim.
(60911 & another embodiment, the isolated placental cells useful ha the methods aiid
¾· compositions described hereto are isolated placental cells tout are one or mote, or all, of CD10+-, CD29+, CD34-, CD38-, CD444, CD45-, CD544, CD904, 5Η2*,$Β3+, S1I4+, SSBA3-. $SEA4%OCT-44~, and ABOpHv where ABC-p to a placenta-specific ABC traitoporter protein (also known as breast cancer resistance protein (BCRP) and as mtioxantrone resistance proteto (MXR)), wherem said isolated placenta cells are obtained by perfusion ofa mammalian, e.g., human, placenta that has been (framed of cord blood and perfused to remove residuat btood. {0092| In another specific embodiment of any Of toe above characteristics, expression of the cellular marker (e.g., duster of differentiation or immunogenic marker) ½ determined by flow cytometry; in another specific embodiment, expression of toe marker to determinedby RT-
PCR.
{00931 Gene pr bfiling confinns that isolated placental cells, and populations of Mated placental cells, are distinguishable from other cells, e.g.„ mesenchymal stem cells, e.g., bone marrow-derived mesenchymal stem cells. Die isolated placental cells described herein can be distinguished from, e.g., mesenchymal stem cells on the Basis of the expression of one or mom genes , the expression Of Which is significantly higher in die isolated placental cells, or in certain isolated umbilical cord stem ce¼ in comparison to bone marrow-derived mesenchymal stem cells. In particular, die isolated placental cells, useful in the methods of treatment provided hereto, can be distinguished from mesenchymal stem cells on toe bask of toe expression of one or more genes, ihe expression of which is significantly Mgher (that is, at least twofold higher) in the isolated placental cells than in an equivalent number of b<w matrow-derived mesenchymal stem cells, wherein toe one or more genes are ACTG2, ADARB 1, AMIG02, ARTS- 1, B4GALT6, BGHE, Cl lor&, CD20O, COMAL COL4A2, CPAADMD, DSC3, D$G2r ELOVL2, 3F2RL1, FU10781, GATA6, GPR126, GPRC5B, HLA-G, 1C AMI, 1ER3, IGFBP7V
ILIA, 1L6, it 18, KRT18, KRT8, LIPG, LRAP, MATNZ, MEST, NFE2L3, NUAKl, PCDH7, PDLIM3, PKP2, RTNl , SERPINB9, ST3GAL6, ST6GALNAC5, SLC12A8, TCF21,TGFB2, VTRZC3M1 2A,m 8 wmbmation of any of the foregoing, when the celts are grown under equivalent conditions. See, e.g., U.S. Patent Application Publication Hb. 2007/0275362, the disclosure of which is incorporated heron by reference in ½ entirety. In certain specific embodiments, said expression of said one ore more genes is detennined, e,g„ by RT-PCR or mtcroainray analysis, using a VI 33-A tnicroarr&y (Asymetrix). In another specific embodiment, Said isolated placental celts express said one or mote genes when cultured for a number of population doublings, e.g., anywhere from about 3 to about 35 population doublings, in a medium comprising DMEM-LG (0.6-, from Gibco); 2% fetal calf serum (e.g,, from Hyclone Labs.); Ix insufin-transfemn-selenium (ITS); ix linoleic acid-bovineserom albumin (LA-BSA); 10-9 M dexamethasone (e.g , from Sigma); 104 M ascorbic acid 2-phosphafo (e.g , from Sigma); epidermal growth factor 10 ng/mL (e.g;, from R&D Systems); and platelet-derived growth ¾tor (PDGF-BB) 10 ng/mL (e.g.. from R&D Systems), lit another specific embodiment, die isolated placental cell-specific m isolated umbilical coni cell-specific gene is CD2Q0.
16094) Specific sequences for these genes can be found in GenBank at accession nos.
NM_001615 (ACTG2X BC065545 (ADARBl), (NMJ 81847 (AM1G02), AY358590 (ARTS- 1), BC0T4884 (B4GALT6), BC008396 (BCHE), BC020196 (Cllorf9), BC03I 103 (CD20OX NM„001845 (CQL4AI ), MMJ301846 (COL4A2), BC052289 (CPA4), BC094758 (DMD), AF293359 (055C3), NMJ>01943 (DSG2X AF338241 (ELOVtJ), AY336105 (F2RLI), KMJH8215 (FUI0781X AY416799 (GATA6), BC075798 (GPR126), NMJU6235 (GPRC5B), AF340038 (1C AM I), BC000844 (1ER3), BC066339 (1GFBP7), 8CQ13142 (ILIA), BTO 19749 (IL6), BC00746I (1LI8), (BC072017) KRTI8, BC075839 (KRT8), BC060825 (LIPGX BC065240 (LRAP), BCOI0444 (MATN2), BC0119O8 (MEST), BC068455 (NFE2L3), NM„014840 (NUAKl), AB006755 (PCDH7), NMJH4476 (PDL1M3), BC! 26199 (PKP-2), BC090862 (RTNI), BC002538 (SERPINB9), BC023312 (ST3GAL6), BC001201 (ST6GALMAC5), BC126160 or BC065328 (SLC12A8XBC025697 (TCF21), BC096235 (TGFB2X BC005046 (VTHX and BCOOSdO! (ZC3H12A) as of March 2008.
{90951 In certain specific embodiments, said isolated placental cells express each of
ACTG2* ADARBl, AM 1602, ARTS-1. B4GALT6, BCHE. CI lori9, CD200, COL4AL COL4A2, CPA4, DMD, DSC3.DSG2, ELOVL2, F2RL1, FU10781, GATA6, GPR126, GPRC5B, HLA-G, fCAM!,lER3, IGFBP7, ILIA, IL6, 1L18, R.RTI8, KRT8, LlPG, LRAP, MAINS, MEST, NFE2L3, NUAKi. PGDH7, PPMM3;PKP2, RtNl, SERPINB9, ST3GAL6, ST6GALNAC5, SLC 12A8, TCF2 i,TGFB2,Vm and ZC3H12A at a detectably higher level tbah an equivalent number of bone marrow-derived mesenchymal stem oils, when the cells am grown under equivalent conditions. in specific embodiments, the placental cells express CD200 and ARTS! (amhopeptidase regulator of type i tumor necrosis factor); ARTS*! and LRAP (leukocyte- derived arginine aminopeptidase); IL6 (mterleukinrti) and TGFR2 (transforming growth factor, beta 2); ILti and KRTl 8 (foaatih 18); 1ER3 (immediate early response 3), WEST (mesoderm specific transcript homofog) and TGF82; CD20O and lER3; CD200 and 1L6; 00200 and KRT18; CD200 and LRAP; CD200 and MEST; CD200 and NEI52L3 (nuclear factor (erythroid- derived 2>Hke 3); or CD20O and TGFB2 at a detectably higher level than an equivalent number of bone marrow-deri ved mesenchymal stem cells (BM-MSCs) wherein said bone marrow- derived mesenchymal stem cells have undergone a number of passages in culture equivalent to the number Of passages said isolated placental cells have undergone. In other specific embodiments, the placental cells express ARTS-1 , CD290, 1L6 and LRAP; ARTS-1 , IL6, TGFB2, IER3, KRT18 and MEST; €D20d, 1ER3, IL6, KRT18, LRAP, MEST, NFE2L3, and TGFB2; ARTS-l, CD200t lER3, IL6. KRT18, LRAP, MEST, NFE2L3, and TGFB2; or IER3, MEST and TGF.B2 at a detectably higher level than an equivalent number of bone marrow- derived mesenchymal stem cells 8M-MSCs, wherein said bone marrow-derived mesenchymal s«em cells have undergone a number of passages in culture equivalent to the number of passages said isolated placental cells have undergone.
160971 Expression of the above-referenced genes can be assessed foy standard techniques.
For example, probes based on the sequence of the gen<$) can be individually selected and constructed by conventional techni ques. Expression of the genes can be assessed, e g., on a microarray comprising probes to one or more of the genes, e g., an Asymetrix GENECR1P® Human Genome U133A 2.0 array, or an Affymetrk GEMECTlIPS Human Genome U 133 Plus 2.0 (Santa Clara, California). Expression of these genes can be assessed even if the sequence for a particular GenBank accession number is amended because probes specific for die amended sequence can readily be generated using well-known standard techniques. j0098f The level of expression of these genes can be used to confirm the identity of a population of isolated placental cells, to identify a population of cells as comprising at least a plurality of isolated placental cells, or foe like. Populations of isolated placental celU, the identity Of which is confirmed, can be clonil, e.g., populations of isolated placental cells expanded from a single isolated platseidal cell, car a mixed population of stem cells, e,g„ a population of cells comprising solely isolated placental cells that are expanded from multiple Isolated placental cells, or a population of cells comprising isolated placental cells, as described herein, and at least one other type of cell.
|β099| The level of expression of these genes can be used to select peculations of isolated placental cells, For example, a population of cells, e,g.. clonaHy-expandedcelis, may be selected if the expression of Ope or more of the genes listed above is significantly higher in a sample from the population tif cells than in an equivalent peculation ofmesenbhymal stem cells. Such selecting cap be of a population from a plurality of isolated placen tal cell populations, fix»» a plurality Of cell populations, the identity of which is not known, etc.
Isolated placental cells can be selected cm foe basis of the level of expression of one ot mote such genes as compared tb the level of expression in said one or more genes in, e.g., a mesenchymal stem cell control, for example, the level of expression in said one or more genes in an equivalent number of bone marrow-derived mesenchymal stem cells. IP one epibodipjent, the level of expression of said one or more genes in a sample comprising an equivalent number of mesenchymal stem cells is used as a control. In another embodiment, the control, for isolated placental cells tested under certain conditions, is a numeric value representing the level of expression of said one or more genes in mesenchymal stem cel ls under said conditions.
1001011 In certain embodiments* foe placental cells (e.g., PDACs) useful in the methods provided herein, do not express CD34, as detected by immunolocalization, after exposure to 1 to 100 ng/mL VEGF for 4 to 2t days, in a specific embodiment, said placental adherent cells are adherent to tissue culture plastic. In anotber specific embodiment, said population of celk induce endothelial cells to form sprouts or tube-like structures when cultured in the presence of an angiogenic factor such as vascular endothelial growth factor (VEGF), epithelial growth factor (EGF), platelet derived growth factor (PDGF) of basic fibroblast growth factor (bFGF), e g. , on a substrate such as MATRZGEL™
(00102} In another aspect, die PDACs provided herein, a population of cells, e.g., a population of PDACs, or a population of cells wherein at least about 50%, 60%, 70%, 80%,
90%, 95% or 98% of cells in said isolated popuktionof cells are PDACs, secrete one or mote* or all. pft/EGF, EOF, 1L-8, MCF-3, FGF2, foltistatm, 0-CSF, EGF, Etf A>?8, GRO, !H MCP-L PDGF-BB, TIMP-2, uPAR, or galectin-l, e.g., into culture medium in which the cell, or cells, are grown, to another embodiment, the PDACs express increased levels ofCD202b^ lL-8 and/or VEGF under hypoxic conditions (e.g,, less than about 5% Q2)compared to hoimoxic conditions (e.g., about 20% or about 21% 02),
160103} to another etnbodijment, any of the PDACS Oi poptitotiohs bf ceilb comprising
PDACs described herein cad cause the formation of sprouts or tuhe-like structures to a population of endothelial cells to contact with said placental derived adherent cells, to a specific embodiment, the PDACs are ccKwhured with human endothelial eeHs, whkh forovsprouts or tubolike structures, or support the formation of endothelial cell sprouts, e.g., when cultured to the presence of extracellular matrix proteins such as collagen type I and IV, and/or angiogenic factors such as vascular endothelial growth factor (VEGF), epithelial growth fector (EOF), platelet derived growth factor (PDGf) or basic fibroblast growth factor (bFGF), e.g., in or on a substrate suchas placental collagen or MATRIGEL™ for at least 4 days. In another embodiment, any of the populations of cells comprising: placental derived adherent cells, described hereto, secrete angiogenic factors such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (bFGF), or toterleukm-8 (IV8) and thereby can induce human endothelial cells to form sprouts or tube-like structures when cultured to the presence of extracellular matrix proteins such as collagen type 1 and IV e.g., in or on a substrate such as placental collagen or MATRIGEL™ j|60104j In another embodiment, any of the above populations of cells comprising placental derived adherent cells (PDACs) secretes angiogenic factors. In specific embodiments, foe population of cells secretes vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet derived growth factor (PDGF), basic fibroblast growth factor (hFGF), and/or mterieukin-S (H>¾). toother specific embodiments, the population of cells comprising PDACs secretes one or more angiogenic factors and thereby induces human endothelial cells to migrate jn an to v¼o wound healing assay, to other specific embodiments, foe population of cells comprising placental derived adherent cells induces maturation, differentiation or proiiferatioft of human endotheiial celts, endothelial progenitors, myocyte or myoblasts. fOOlOSj The isolated placental cells described hereto display the above characteristics (e g,, combinations of cell surface markers and/or gene expression profiles) in primary culture, or during proliferation in medium comprising, e g., DMEM-LG (Gibed), 2% fetal calf sentm (PCS) (Hyclone Laboratories), lx insulimnansferrm-sdenium (ITS), lx tenolenic-acidrbovme^semm- albumin (LA-BSA), 10-9 M dexamethasone (Sigma), 10-4M ascotbic acid 2-phosphate (Sigma), epidermal growth factor (EGFUOhg/nil (k&D Systems), platelet derived-growth factor (PDC5F- BB) iOrtg/m) (R&D Systems), and jOOU penicillm/lOOOU streptomycin.
(001061 In certain embodiments of any of the placental cells disclosed herein, the cells are human. I» certain embodiments of any of the placental cells disclosed herein, the cellular marker characteristics of gene expression characteristics ate human markers or human genes.
(001071 In another specific embodiment of said isolated placeptal ceUsorpopulatioas of cells comprising the isolated placental cells, said cells or population have been expanded, W example, passaged at least, about, or no more than, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times, or more, or proliferated fdr at least, about, or nb mote than, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39,40,41,42, 43, 44, 45, 46, 47,48, 49 or 50 population doublings.
In another specific embodiment of said isolated placental cells or populations of cells comprising the isolated placental cells, said cells or population are primary isolates, in another specific embodiment of the isolated placental cells, or populationsof celiscomprising isolated placental cells, that are disclosed herein, said isolated placental cells are fetal in origin (that is, have the fetid genotype).
In certain embodiments, said isolated placental cells do not differentiate during culturing in growth medium, ie„ medium formulated to promote proliferation, e.g., during proliferation in growth medium. In another specific embodiment, said isolated placental cells do not require a feeder layer tit order fb proliferate, in another specific embodiment, said isolated placental celts do not differentiate in culture in the absence of a feeder layer, solely because of the lack of a feeder cell layer.
(00109} In another embodiment, cells usefiil in the methods and compositions described herein are isolated placental cells, wherein a plutatiiy of said isolated placental cells are positive ibr aldehyde dehydrogenase (ALDH), as assessed by an aldehyde dehydrogenase activity assay Such assays are known in the art (see, eg.4 Bostian and Betts, Biochemu J., 173, 787, ( 1978»; In a specific embodiment, said ALDH assay nses Aldefluor® (Aldagen, Inc., Ashland, Oregon) as a marker of aldehyde dehydrogenase activity, lit a specific embodiment, said plurality is between about 3% and about 25% of cells in said population of cells. In another embodiment, provided herein is a population of isolated umbilical coed cells, e-g·. multipotent isolated umbilical cord cells, wherein a plurality of said isolated umbilical cord celts are positive for aldehyde dehydrogenase, as assessed byan aldehyde dehydrogenase activity assay dial uses Aldefluor® as an indicatotofaldehyde dehydrogenase activity. In a specific embodiment said plurality is between about 3% andabout 25% Of cells in said population of cells. In andtheranbodiment, said population of isolated placental cells or isolated umbilical cord cells shows at least three- fold, or ar least fi ve-fold, higher ALDH activity than a population of bone marrow-derived mesenchymal shun cells having about the same number of cells and cultured under the same conditions.
160119) In certain embodiments of any of the populations of cells comprising the isolated placental cells described herein, the placental cells in $&d populations of cells are substantially free of cells having a maternal genotype; e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 30%, 35%, 00%, 95%, 98% or 99% of the placental cells in said population have a fetal genotype. In certain otlw embodtments of any of the populations of cells comprising me Isolated placental cells describedherein, the populations of cells comprising said placental cells are substantially free of cells having a maternal genotype; e.g,, at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% of the cells In said population have a fetal genotype.
{601111 In a specific embodimeOtofaoy of the above isolated ptacental cells or cells populations ofisolated placental cells, the karyotype of the cells, or at least about 95% or about 99% of the cells in said population, is normal lu another specific embodiment of any of the above placental cells or cell populations, the cells, or cells in the population of cells, are non- maternal in Origin.
190112) Isolated placental cells, or populations of isolated placental cells, bearing doy of the above combuiatiohs Of markets, can be combined in any ratio. Any two or more of the above isolated placental cell populations can be combined to form an isolated placental cell population. For example, an population of isolated placental cells caft comprise a first population of isolated placental cells defined by one pf the marker combinations described above, and a second population of isolated placental cells defined by another of the marker combinations described above, wheteiti said Hist atidsecond populations are combined in a ratio of about 1 ;99, 2:98, ¾97, 4:96, 5:95, 10:90,20:80, 30:70, 40160, 50:50, 6θϊ4θ; 70:30r 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, or about 99.1, l« tike fashion, any three, four, five or more of tlie a^ove-described isolated placental celts or isolated placental cells populations cap be combmed
(001131 Isolated placental cells useful in the methods and compositions described herein cab be obtained, e.g., by disruption of placental tissue, with or without etwymatic digestion (See Section 4.2.3) or perfusion (see Section 4.2.4). For example, populations Of isolated placental cells can be produced according to a method comprismg perfhsing a mammalian placesia that has been drained of cord blood and perfused to remove residual blood; perfusing said placenta with a perfusion solution; and collecting said perfusion solution, wherein said perfusion solution after perfbsio» comprises a population of placental cells that coinprises isolated placental Cells; and isolating a plurality of said isdlated placemal cells from said population of cells. In a specific embodiment, die perfusion solution is passed through both the umbilical vein and umbilical arteries and collected after it exudes from the placenta, In another specific embodiment, die perSsion solution is passed through the Umbilical veiiiabd collected from the Umbilical arteries, or passed through die umbiHcaiurtetiesand collected from the umbilical vein.
(00114] In various embodiments, the isolated placental cells, contained within a population of cells obtained from perfusion of a: placenta, are at least 50%, 60%, 70%, 80%,
90%, 95%, 99% or at least 99.5% of said population of placental cells, in another specific embodiment, the isolated placental cells collected by perfusion comprise fetal and maternal cells. In atrother specific embodiment, the isolated placental cells collected by perfusion are at least 50%, 60%.70%, 89%, 90%, 95%, 99% or at least 99.5% fbtal cells.
(001151 In another specific embodiment, provided herein is a composition cotaprismg a population of the isolated placental ce¼ as described herein, collected by perfusion, wherein said composition comprises at least a portion of the perfusion Solution used to collect the Isolated placentalcells-
1001161 Isolated populations of the isolated placental cells described herein can be produced by digesting placental tissue With a tissue-disrupting enzyme to obtain a population of placental cells comprising the cells, and isolating, or substantially isolating, a plurality of the placental cells from the remainder Of said placental cells. The Whole, Or any part of, the placenta can be digested to obtain the isolated placental ©ells described herein. In specific embodiments, tor example, said placental tissue can be a whole placenta, an ammotic membrane, chorion, a combination of antoion and chorion, or a combination of any of the foregoing. In other specific embodiment, the tissue^isrupting enzyme is trypsin or coliagenase. in Various embodiments, the isolated placental cells, contained within a population of cells obtained from digesting a placenta, are at least 50%, 60%, 70%, 80%, 90%, 95%, 99% oral least 99:5% of said population of placental celts.
1001171 Theisotated populations of placentalceUs describedabove* and populations of isolated placental cells generally, can comprise about, at least, or no more than l x L0\ 3 x 10\ 5
X 103, 1 X 10*.3 x lOVSx ID4, 1 X io5, 3 x W\ 5 x 105, 1 x 10®, 3 x IO6, 5 x 106, t * ID7, 3 x SO7, 5 k ] 07, 1 X itf, 3 X 10s, 5 x 10s, I x 109, 5 x IO9, or 1 k 10i0 isolated placental cells (e.g., as part of a pharmaceutical composition comprising placental stem cells) or between about: 1 x 10* to 3 x 103, 3 x 1(P to 5 x 10*, 5 x 10s to I x 104, 1 χ 10* to 3 x 104, 3 x K^to 5 x 104, $ x 104 to 1 x 10s,
I x 10* to 3 X 10s, , 3 x itfto 5 x !O5, 5 x 10* to l x 10*, t x 10* to 3 x IO6, 3 X 10%ο 5 x Id6, 5 x 106 to 1 x 107, 1 x 107to 3 x 107, 3 x IQ7 to 5 X 107, 5 X 10? to 1 x 108, 1 x 10* to 3 x 10®, 3 X 10s to 5 x 10s, 5 x 10s to 1 x IO9, 1 x 10* to 5 x 109, or 5 x 109 to 1 x 10<9 isolated placental cells (e.g., as part of a pharmaceutical composition comprising placental stem celts). Populations of isolated placental cells useful in the methods of treatment described herein comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, Of 99% viable isolated placental cells, &g., as deterntined by, e.g., trypan blue exclusion,
4.2 METHODS OF OBTAINING ISOLATED PLACENTAL CELLS
4-2,1 M Qflwtiwi, C.ompff^riop
1001181 Further provided herein am methods of collecting and isolating p lacental cells,
&g., the isolated placetital cejQs described in Section 4, i, above. Generally, such ceiis are obtained ¾m a mammalian placenta using a physiologlcally-acceptable splution, e-.g., a cell collection composition. Am exemplary cell collection composition is described in detail in related U S. Patent Application Publication No. 2007/0190042, entitled “Improved Medium tor Collecting Placental Stem Cells and Preserving Organs,” the disclosure of which is incorporated herein by reference in its entirety {90119] The cell cotiebtien composition can comprise any physiologi<&lly-aeceptable solution suitable for the collection and/or culture of cells, e.g., the isolated placental cells described hereto, for example, a saitoe solution (e.g., phosphate-buffered saline, Kteb’s solution, modified Kreb’s solution, Eagle’s solution, 0,9% Na0. etc ), a culture medium (e.g., DMEM. H.DMEM, etc,), and the like,
100126] The ceil collection composition can comprise One or more components that tend to preserve isolated placental cells, that is, prevent the isolated placental cells from dying, or delay the death of the isolated placental cells, reduce the number of isolated placental cells m a population Of cells that die, or the like, from the time of collection to the time of cuttaring. Such components can be, e.g., an apoptosis inhibitor (e.g„ a caspase inhibitor or JNK inhibitor); a vasodilator (e.g,, magnesium sulfate, an anttoypertensive drug, atrial natriuretic peptide (ANP), adrenocorticotropitt, corticotropin-ieleosing hormone, sodium nitropmsside, hydralazine, adenosine triphosphate, adenosine, indomethacin or magnesium sul&te, aphosphodiesterase inhibitor, etc.); a necrosis inhibitor (e.g., 241H-lnddh3«yl)-3*pentylami00-maleimide, pyrrolidine dithjocarbamate, or clonazepam); a TNF-a inhibitor', and/or an oxygen-carrying perfiuorocarbon (e.g„ perfluorooctyl bromide, perfluorodecyl bromide, etc.},
160121] The cell collection composition can comprise one or more tissue-degrading enzymes, e.g., a metallcptotease, a Serine pmtease, a neutral protease, an RNase, m i DNase, or the like. Such enzymes include, but are not limited to, collagenases (e.g., collagenase l, II, III or IV, a collagenase from Clostridium histolytiawuetc.}; dispose, thermoiysin, ejastase, trypsin, UBERASE, hyaiuronidase, arid the like. f001221 The cell collection composition can comprise a bacteriocidally or bacteriostaticaUy effective amount ofan antibiotic In certain non-limiting embodiments, the antibiotic is a macrolide (e.g., tobmmycm), a cephalosporin (e.g., cephalexin, cephractine, cefriroxime, celprozil, cefaclor, cefixime dr cefadroxti), a clarithromycin, an erythromycin, a penicillin (e.g.t penicillin V) or a quiMone (eg.. Ofloxacin, ciprofloxacin or norfloxacin), a tetracycline, a streptomycin, etc. In a particular embodiment, the antibiotic is acti ve against Gram(4) and/or Gram(-) bacteria, e g., Pseudomonas aeruginosa, Staphylococcus auteus, and the like. In one embodiment, the antibiotic is gentamycin, e.g., about O:O05% to about 0,01% fw/V) in culftue medium {00123 j The ceil collection composition can also comprise one or more of the following compounds: adenosine (about I mM to about 50 mM); D-giucose (about 20 niM to about 100 mM); magnesium ions (about I mM to about 50 mM); a macromolecule of molecular weight greater man 20,000 dalfons, in ode embodiment praseitt in an amount sufficient to maintain endothelial integrity and cellular viability (e.g-, a synthetic or naturally occurring colloid, a polysaccharide such as dextran or a polyethylene glycol present at about 25 g/1 to about 100 g/1, or about 4ft g/l to about 60 g/l); an antioxidant (e.g,, botylatcd hydroxyaniSOle, butylated hydfOxytoluene, glutathione, vitamiii C or vitamin E present at about 25 μΜ to about 100 μΜ); a reducing agent (e. g,, N-acetylcysteine present at about 0.1 mM to about 5 mM); an agent that prevents calcium entry into cells (e.g., verapamil present at about '2 μΜ to about 25 pM); nitroglycerin (e.g., about 0.05 g/L to about 0.2 g/L); an anticoagulant, in one embodiment, present in an amount sufficient to help prevent clotting of residual blood (e.g., heparin dr hirudin present at a concentration of about lOOO unitsd to about 100,000 units/!); or an amiloride cpniauiing compound (e.g., amiltiride, ethyl isopropyl amiloride, hexamethylene amiloride, dimethyl amiloride or isobutyl amiloride present at about 1.0 μΜ to about 5 μΜ).
42.2
{001241 Generally, a human placenta is recovered shortly after its expulsion after birth, in a pteferred embodiment, the placenta is recovered from a patient after informed consent and after a complete medical history of the patient is taken and is associated with the placenta. Preferably, Hie medical history continues after delivery. Such a medical history can be used to coordinate subsequent use of me placenta or the isolated placental cells harvested therefrom. For example, isolated human placental cells can be used, in light Of the medical history , for personalized medicine for die infant associated with the placenta, or for parents, siblings or other relatives of the infant
{00125} Prior to recovery of isolated placental cells, the umbilical cord blood and phcental blood are preferably removed. In certain embodiments, after delivery, the cord blood m the placenta is recovered. The placenta can be subjected to a conveotional cord blood recovery process, Typically a needle dr cannula is used, with the aid of gravity, to exsanguinate tire placenta (see, e.g., Anderson, U.S. PaienfNo. 5,372,581; Hessel et al., U,S, Patent No,
5,415,665), The needle or cannula ½ usually placed in the umbilical vein and the placenta can be gentlymassaged toaid in draining cord blcxKl from the placenta. Such cord blood recovery may be performed commercially, e,g., LifeBank USA, Cedar Knolls, Hi. Preferably, the placenta is gravity drained without further manipulation so as to minimize tissue disruption during cord blood recovery.
{00126} Typically, a placenta is transported frorothe delivery or birthing room to another location, eg,, a laboratory, for recovery of cord blood and collection of stem cells by, eg., perfusion or tissue dissociation. The placenta is preferably transported in a sterile, thermally insulated transport device (maintaining the temperature of the placenta between 20-28¾C), for example, by placing the placenta, with clamped proximal umbilical cord, in a sterile zip-lock plastic bag, which is then placed ½ un insulated container. In another embodiment, the placenta is transported bin cord blood collection kit substantially as described in pending United States Patent No.7, 147,62b, die disclosure of Which is imrotporated by reference herein. Preferably, the placenta is delivered to foe laboratory four to twenty-four hours following delivery in certain embodiments, foe proximal umbilical cord is clamped, preferably within 4-5 cm (centimeter) of foe insertion into the placental disc prior to cord blood recovery, In other embodiments, foe proximal umbilical cord ¼ clamped after cord blood recovery but prior to further processing of foe placenta,
{00127} The placenta, prior to cell collection, can be stored trader sterile conditions trad at either room temperature or at a temperature of 5°C to 258C. The placenta may be stored for a period Of for a period of four to twenty-four hours, up to forty-tight horns, or longer than forty eight hours, prior to perfusing the placetita to remove any residual cord bibod. In one embodiment, foe placenta is harvested from between about zero hours to about two hours post- expulsion. The placenta is preferably stored in an anticoagulant solution at a temperature of 5°C to 25*C. Suitable anticoagulant solutions are well known in foe art. For example* a solution Of heparin or Warforip sodium can be used. In a preferred embodiment, the anticoagulant solution comprises a solution of heparin (e.g,, 1% w/w in 1:1000 solution) . The exsanguinated placenta is preferably stored for no more than 36 hours before placental cells are collected- f 00128) The mammalian placenta or a part thereof, once collected and preparedgeneraily as above» can be treated in any art-known manner, can be perfused or disrupted. <r,g., digested with one or more tissue-fosrupting enzymes, to obtain isolated placental cells. 4.23 Physical Disruotioif and Enzymatic Digestion of H ΕΪΗΜί,.
|0O129| in one embodiment, stem cells are collected from a mammalian placenta by physical disruption of part of all Of the organ. For example, the placenta, or a portion thereof, may be,e,g.„ crusted, sheared, minced, diced, chopped, macerated or the like. The tissue can then be cultured to obtain a population of isolated placental cells. Typically, the placental tissue |S disrupted using, e.g., culture medium, asaime solution, or a stem cell collection.
(601301 The placenta can be dissected into components prior to physical disruption and/or enzymatic digestion and stem cell recovery. Isolated placental cells can be obtained from all or a portion qf ite amntotic membrane^chorion, umWcal cord, placental cotyledons, or any combination thereof, including from a whole placenta. Preferably, isolated placental cells are Obtained from placental tissue comprising amnion and chorion. Typically, isolated placental cells can be obtained by disruption Of a small block of placental tissue, e.g., a block of placental tissue that is about 1, 2, 3, 4» 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900 of about 1000 cubic millimeters in volume. Any method of physical disruption can be used, provided that the method of disruption leaves a plurality, mote preferably a majority, and more preferably at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% of the cells in said organ viable, as determined by, e.g., trypan blue exclusion.
(001311 The isolated adherent placental cells can generally be collected from a placenta, or portion thereof, at any time within about the first three days post-expulsion, but preferably between about 8 hours and about 18 hours post-expulsion.
(00132} to a specific embodiment, the disrupted tissue is cultured in tissue culture medium suitable for the proliferation of isolated placental cells.
(661331 In another specific embodiment, isolated placental cells are collected by physical disruption of placental tissue, whereto the physical disruption includes enzymatic digestion, which can be accomplished by use of one or more tissue-digesting enzymes. The placenta, or a portion thereof, may also be physically disrupted and digested With rate or mote enzymes, and the resulting material then immersed to, or mixed into, a cell collection composition.
(60134} A preferred cell collection competition comprises one or more tmue-disniptive enzyme(s). Enzymes that can be used to disrupt placenta tissue include papain, deoxyribonticleases^serme proteases, suchas trypsin, ohymbtrypsin, collagenase, dtspase or eiastase. Smite proteases may be inhibited by alpha Z microglobulm in serum and therefore the medium used for digestion Is usually serum-foee. BDTA and PNase are commonly used in enzyme digestion procedures to increase the efficiency of celt recovery. The digestate is preferably diluted sous to avoid trapping cells within the viscous digest
(00135 j ½y combination of tissue digestion enzymes can be used. Typical concentrations For digestion using trypsin include, 0:1% to about 2% trypsin, e.g,. about 0.25% trypsin. Proteases can be used in combination, that is, two or more proteases in the same digestion reaction, or can be used sequentially in order to liberate placental cells, e.g., placental
X stem cells and placental multipotent cells. For example, m due embodiment, a placenta, or part Seiedf. is digested first with ad appropriate amount of cotlagenase t at about 1 to about 2fng/tnl for, e.g,, 30 minutes, followed by digestion with trypsin, at a concentration of about 0.25%, for, e.g., 10 minutes, at 37*C. Serine proteases are preferably wd consecutively following use of other enzymes.
(001361 in another embodiment, the tissue cab further be disrupted by the addition of a chelator, e.g , ethylene glycol bis(2-ammoethyf efoer)-N,N,N<MMetraaceticacid (EOTA) or ethylenediaminetetraacetic acid (EDTA) to the stem cell collection composition comprising the stem cells, or to a solution ih Which the tissue is disrupted and/or digestetiprior to isolation df the stem cells with the stem cell collection composition.
1001371 Following digestion, the digestate is washed, for example, three timeswifo culture medium, and the washed cells are seeded into culture flasks. The cells are then isolated by differential adherence, and characterized for, e.g., viability, cell surface markers, differentiation, and foe like.
(00138| I t will be appreciated that where an entire placenta, or portion of a placenta comprising both fetal and maternal cells (for example, where foe portion Of the placenta comprises foe chorion or cotyledons), the placental cells isolated can comprise a mix df placental cells derived from both fetal and maternal sources. Where a portion of foe placenta that comprises Mb, or a negligible number of, maternal cells (for example, amnion), foe placental cells isolated therefrom will comprise almost exclusively fetal placental cells (that is, placental cells having foe genotype of the fetus). )0O139) Placental cells, e.g., the placental calls described in Section 4.1, above, can be isolated from disrupted placental tissue by differential trypsihization {see Section 4.2.5, below) followed by culture in one or mote new ctilttite tontainers in fresh proliferation medium, optionally folJowedby a seccmddifferential ttypsinization step.
414 Placental Perfusion
1001401 Placental cells,*#., the placental cells described in Section 4.1, above, can also be obtained by perfusion of the mammalian placenta. Methods of perfusing mammalian placenta to obtain placenta! Cells are disclosed, e.g., iu Hariri, U.S. PaietuNos, 7,045,148 and 7,255,729, ½ Li.$. Patent Application Publication Nos.2007/0275362 and 2007/0190042, the disclosures of each of which are incorporated herein by reference id their entireties;
(80141! Placental cells can be collected by perfUsion, e.g., through the placental vasculature, using, e.g., a cell collection composition as a perfusion solution. In one embodiment, a mammalian placenta is perfused by passage of perfusion solution through either or both of the umbilical artery and umbilical vein. The flow of perfusion solution through the placenta may be accomplished using, e.g., gravity flow into the placenta. Preferably, the perfusion solution is forcedtbrough the placenta using 8 pump, e.g.,a peristaltic pump. The umbilical vein can be, e.g., cannulated with a cannula, e.g., a TEFLON® or plastic cannula, that is connected b a sterile connection apparatus, such as sterile tubing. Hie sterile connection apparatus is connected to a perfusion manifold. j 00142) In preparation fbr perfusion, tiie placenta is preferably oriented (e.g., suspended) in such a manner that the umbihcal artery and umbilical vein are located at the highest point of the placenta. The placenta can be perfused by passage of a perfusion fluid through tie placental vasculature and surrounding tissue. The placenta can also be perfused by passage of a perfhsion fluid into the umbilical vein and collection from tie umbilical arteries* or passage of a perfusion fluid into the umbilical arteries and collection from the umbilical vein.
(00143) tu one embodiment» for example, the umbilical artery arid the umbilical vein are counected simuhaneqitoly, e.g., to a pipette that is connected via a flexible connector to a reservoir of tie perfusion solution» The perfusion solution is passed into the umbilical vein and artery. The perfusion solution exudes from and/or passes through the walls of tie blood vessels into tie surrounding tissues of the placenta, and is collected in a suitable open vessel from the surihee of foe placenta that was attached to foe uterus of the mother during gestation. The perfusion solution may Slso be introduced through the umbilical cord opening and allowed to flow or percolate out of openings in foe wall of the placenta which interfacedwith theroaternal Uterine wall. Placental cells foat are collected by this method, which can be referred to as a “pan” method# ate typically a mixture of fetid and maternal cells#
(00144$ In another embodiment, the perfusion solution is passed through foe umbilical veins and collected from foe umbilical artery, or is passed through foe umbilical artery and collected from foe umbilical veins. Placental cells collected by this method, which CM be referred to as a “closed circuit” method, are typically almost exclusively fetal.
$00145$ It will be appreciated that perfusion using foe pan method, that is. Whereby perfusate is collected after it has exuded from the maternal stde offoe p!acehta, results in a mix of fetal and maternal cells. As a result, foe cells collected by this method can comprise a mixed population of placental cells, e.g#, placental stem cells or placental multipotem cells, of both fetal and maternal origin. In contrast, perfusion solely through foe placental vasculature In the closed circuit method, whereby perfusion fluid is passed through one or two placental vessels and is collected solely through foe remaining vessels), results in foe collection of a population of placental cells almost exclusively of fetal origin.
100146$ The closed circuit perfusion method can, id one embodiment, be performed as follows. A post»partum placenta is obtained within about 48 hours after birth. The umbilical cord is clamped and cut above foe clamp. The umbilical cord cah be discarded or can processed to recover, e*g.,«mbiUcai cord stem ce¾ and/or tp process foe umbilical cord membrane for foe production of a biomaterial. The amniotic membrane can be retained during perfosion, or can be separated from foe chorion, e.g., using blunt dissection with the fingers. If the amniotic membrane & separated from foe chorion prior to perfusion, k can be, e g., discarded, or processed, e.g., to obtain stem cells by enzymatic digestion, or to produce,, e.g,, an amniotic membrane biomaterial, e g,, foe bidmaterial described in U S. Application Publication No. 2004/0048796, the disclosure of which is ineorpofatedby reference herein in its entirety. After cleaaiftg the placenta of all visible blood clots and residual blood, e.g„ using sterile gauze, the umbilical cord vessels are exposed, e.g,, by partially cutting the umbilical cord membrane to expose a cross-section of the cord. The vessels are Identified, and opened; eg., by advancing a closed alligator clamp thtoughthe cut end of each vessel Theapparatus, eg., plastic tubing connected to a perfusion device or peristaltic pump, is then inserted into each of the placental arteries. The pump cait be any pttmp sttitable for the purpose, e g., a peristaltic pump. Plastic tubing, connected to a sterile collection reservoir, e g., a blood bag such as a 250 mL collection bag, is fee» inserted into fee placental vein, Alternatively, fee tubing connected to fee pump is inserted into fee placental vein, and «fees to a collection reservoir(s) are inserted ifeo one or both of the placental arteries. The placenta is then perfused with a volume of perfusion solution, e.g^, about 750 ml ofpetfeston solution. Cells in the perfusate are then collected, e.g,, by centrifugation. In certain embodiments, the placenta is periused with perfuSion sohmon, e.g., 100-300 mL perfesion solution, to remove residual blood prior to perfusion to collect placental cells, e.g., placental stem cells and/or placental muitipotent cells. In another embodiment, the placenta is nbt perfused wife perfusion solution to remove residual blood prior to perfusion to col lect placental cells .
(001471 In one embodiment, the proximal umbilical cord is damped during perfusion, and mom preferably, is damped Within 4-5 cm (centimeter) of fee cord's insertion into fee placental disc.
(00148} The first collection Of perfusion fluid from a mammalian placenta during the exsanguinattoh process is generally colored wife residual red blood cells of fee cord blood and/or placental blood. The perfusion fluid becomes more colorless as perfiision proceeds and the residual cord blood cells- ate washed out oftheplacenta. Generally flrotn 30 to 100 ml (milliliter) of perfusion fluid is adequate to initially exsanguinate the placenta, but more or less perfusion fluid may be used depending On fee observed results,
160149} The volume of perfusion liquid used to isolate placental cells may vary depending upon fee number of cells to be collected, fee size of the placenta, fee number of collections to be made from a single placenta, etc, h various embodiments, fee volume of perfusion liquid may be from 50 mL to SOOO mL, SO ntLto 4000 mL, 50 mL to 3OO0 ntL, 100 mL to 2000 mL, 250 mL to 2000 ml, SOO mL to 2000 mL, or 750 mL to 2000 mL. Typically, the placenta is perfused wife 700-800 mL of perfusfeti liquid folldwmg exSatiguination.
(001501 The placenta can be perfused a plurality of times over fee course of several hours or several days. Where, the placenta is to be perfused a plurality of times, it may be maintained or cuitwred under aseptic conditions in a container or other suitable vessel, and perfused with the cell collection composition, or a standard perfusion solution (e.g., a normal saline solution such as phosphate buffered salhe f ‘PBS”)) with or Without an anticoagulant (e g., heparin, warfarin sodium, coumarin, btshydroxycmimarin), and/or with or without ait antimicrobial agent (e.g., β* mercaptoethanol (0,1 mM); antibiotics such as streptomycin (e.g., at 40-100 pg/tul), penicillin
(e.g., at 40L)/ifti|, amphotericin B (e.g., at 0.5 pg/rUl). In one embodiment, an isolated placenta is
: maintained or cultured tin a period of time without collecting the perfusate, such that the placentitis maintained Of Cultoredfor 1, 2, 3.4, 5, 6, 7, 8, 9, 10, Π, 12, 13» 14, 15; lb, 17» 18, 19, 20, 21, 22, 23, or 24 hours, or 2 or 3 or mtire days before perfusion and collection Of perfusate. The perfused placenta can be maituaihed for one or more additional timrfs), e,g., t, 2, 3, 4, 5, 6, 7, 8» 9, 10, 11, 12, 13, 14, 15, 16, 17, i 8, 19, 20, 21, 22, 23, 24 or more hours, and perfused a second time with, e,g„ 700-800 tuL perfusion fluid. The placenta can be perfused 1 , 2, 3, 4, 5 or more times, tor example, once every 1 , 2, 3 , 4, 5 or 6 hours. In a preferred embodiment, perfusion of the placenta and collection of perfusion solution, e.g., Cell collection composition, is repeated until the number of recovered nucleated cells falls below 100 ceIJs/mL The perfusates at different time points can be further processed individually to recover time-dependent populations Of cells, e g,, Stem cells: Perfusates from dirfereni time points can also be pooled. In a preferred embodiment, placental cells are collected at a time or times between about 8 hours and about I S hows posr-expulsiom
1001511 Perfusion preferably results in the collection of significantly more placental cells than rite number obtainable from a mammalian placenta not perfused with stud solution, and not otherwise seated to obtain placental cells (e.g , fey tissue disruption, e.g.t enzymatic digestion).
In this context, ‘‘significantly more” means a» least 10% more. Perfusion yields significantly mmre placental cells than, e.g.* the number of placental cells isolatable from culture mediant in which a placenta, or portion thereof has been cultured.
J0Q152J Placental cells can be isolated from placenta by perfusion with a solution comprising one car mote proteases pr other tissuenfistuptive enzymes. In a specific embodiment, a placenta or portion thereof (e.g., amniotic membrane, amnion and chorion, placental lobule or cotyledon* Umbilical cord, or combination of any of the foregoing) is brought to 25-37T, and is incubated with one or more tissne-disraptive enzymes in 200 mL ofa culture medium for 30 minutes. Cells frdm toe perfusate are collected, brought to 4°C, and Washed with a cold inhibitor mix comprising 5 mM EDTA.2 mM difoiothreitol and 2 mM bete-merCaptoefoanol. The placental cells are washed after several minutes with a cold (e^*, 4°C) stem cell collection composition.
4,2*5 Isolation. Sorting, and Characterization of Placental Cells
(00153) The isolated placental cells, the cells described *tt Section 4, 1 * above, whether obtained by perfusion or physiol disruption, e.g,, by enzymatic digestion, can initially be purified from (/.e.,be isolated from) other cells by Ficoll gradient centrifugation. Such centrifugation can follow any standard protocol for centrifugation speed, etc. In one embodiment, for example, cells hoisted from the placenta areiecovered frofo perfttsate by ttentrifugatioh at 5000 it g for 15 minutes at room temperature, which separates cells from, e.g., c½tamtnating debris and platelets* In another embodiment, placental perfusate is concentrated to about 200 ml, gently layered over ficoll, and centrifuged at about 11 P0 x g for 20 minutes at 22% and the low-density interfile layer of cells is collected for further processing. f 00154) Cell pellets can be resuspended in fresh stem cell collection composition, or a medium suitable for cell maintenance^ e.g., stem cell maintenance, for example, 1MDM serum- free medium containing 2U/ml heparin ahd 2 mM EDTA (GibcoBRL, NY). Die total mononuclear celt fraction can be isolated, e.g., using Lymphoprep (Nycomed Pharma, Oslo, Norway) according $0 the manufacturer's recommended procedure. ftiOlSS) Placental cells obtained by perfusion or digestion can. for example, be further; or initially, isolated by differential trypsinization using, e.g., a solution of 0.05% trypsin with 0.2% EDTA (Sigma, St Louis MO); Differential frypsidizatioti is possible because the isolated placenta! celts, which are tissue culture plastic-adherent, typically detach from the plastic surfaces within about five itimutes whereas other adherent populations typically requite more than 20-30 minutes incubation. The detached placental Celts can be harvested following trypsinization and trypsin neutralization, using, e.g., Trypsin Neutralizing Solution (TNS, Cambrexl Εή one embodiment of ½iation of adherent cells, aliquots of, for example, about 5- 10 x W cells are placed in each of several T-75 flasks, preferably fibronectin-coated T75 flasks. In such an embodiment, foe cells can he cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) (Ourabrex), and placed in a tissue culture incubator (37% 5% CD2). After 10 to 15 days, non-adherent cells are remoVed frOm the flasks by waslihg with PBS. The PBS is then replaced by MSCGM Flasks are preferably exaromeddaily for foe presence of various adherent cell types and m particular, for identification and expansion of clusters of fibroblastoid cells .
{00156} The number and type of cells collected from a mammalian placenta can he monitored* for example, by measuring changes in morphology and cell surface markers using standard cell detection techniques such as flcw cyfotnetiy, cell sorting, immimocytochemistry (e.g., staining with tissue specific or cell-marker specific antibodies) fluorescence activated cell sorting (FACS), magnetic activated ceil sorting (MACS), by examination of the morphology of cells using light or confocal microscopy, and/or by measuring changes in gene expression using techniques well known in the ait, such as ICR and gene expression profiling. These techniques can be used, too, to identify cells that ate positive for one or mote particular markers. For example; using antibodies to CD34, one cap determine, using the techniques above, whether a Cell comprises a detectable amount Of CQ34; if so, the cell is CD34+: Likewise, if a cell produces enough OCT-d RNA to be detectable by RT-FCR, or significantly more OCT-4 RNA than an adult cell, foe cell ¼ 0CT-4+. Antibodies to cell surface markers (e,g.y CD markers snch as CD34) and the sequence ofstem cell-specific genes, such as OCT-4, are well-known in the art-
{00*57} Placental cells, particularly cells that have been isolated by Ficoll separation, differential adherence, or a combination of both, may be sorted using a fluorescence activated cell sorter (FACS), Fluorescence activated celt sorting (FACS) is a well-known method for separating particles, including cells* based an the fluorescent properties of the particles (Kamarch, 1987, Methods Enzymol, ΐ51;150-¼5). Laser excitation of fluorescent moieties in the individual particles results in a small electrical charge allowing electromagnetic separation of positive and negative particles from a mixture. In one embodiment, cell SiufacO marker-specific antibodies or ligands are labeled with distinct fluorescent labels. Cells are processed through the cell sorter, allin½g separation of cells based on their ability to bind to foe antibodies used, FACS sorted particles may be directly deposited into individual wells of 96-well or 384-well plateS to facilitate separation and cloning.
{00158} tn one sorting scheme, cells from placenta, e.g., PDACs are sorted on the basis of expression of one or moreof the markers CD34, CD38, Cb44, CD45, CTO, CPJOS, OCT-4 and/or HiA-G. This cab be accomplished it* coutieetion with procedurestb select such cells on t¾ basis oftheir adherence properties in culture- For example, tissue culture plastic adherence selection can be accomplished before or after sorting: on toe basis of market expression, to one embodiment for example, cells are itorted first On the basis of their expression of CD34; CD34- cells are retained, and CD34- cells that are additionally CD20CM and HLA-G- are Separated from all other CD34 - cells, to another embodiment, cells ftom placenta ate sorted based bn their expression of markers CD2QO and/or HLA-G; for example, cells displaying CD20G and lacking BLAHS are isolated tor further use. Cells that express, e.g„ CD200 and/or lack, e.g., HLA-G can. in a specific embodiment, be further sorted based on their expression of CD73 and/or CD 105, or epitopes recognized by antibodies SH2, SH3 or SH4, or lack of expression bfPD34; CD38 or GD45. For example, in another embodiment, placental cells ate sorted by expression, or lack thereof, of CD200, HLA-& GD?3, CD! 05, CD34, GD38 and CD45, and placenta) cells that are CD200+, HLA-G-, CD73+. CD105+, CD34-, CD38- and CD45- are isolated ftom otoer placental cells tot further use;
(60159) I» specific embodiments of any of the above embodiments of sorted placental bells, at least 50%, 60%, 70%, 80%, 90% or 95% offitecellsih a cell population remaining after sotting are said isolated placental cells. Placental cells can be sorted by one or mom of any of the markers described in Section 4, 1, above.
160166) In a specific embodiment, for example, placental cells that are (1) adherent to tissue culture plastic, and (2) CJDtlOz, CD34- and CD 105f m sotted ftotti (i.e., isolated from) other placental cells, to another Specific embodiment, placental cells that are (1 ) adherent to tissue culture plastic, and (2) CD'IO*·, CD34-, CD 105+ and CD200+ are spirted from (i.e., isolated from) other placental cells. In another specific embodiment, placental cells that are ( 1 ) adherent to tissue culture plastic; and <2) CtN 0*, CD34-, CD45-, C»9<H% CD165+ and CD2P04< are sorted from (i.e., isolated from) other placental cells.
160161) With respect to nucleotide sequenceybased detection ofplaeental dells, sequences for the markers listed herein are madily available in pubticiy-available databases such as GenBank or EMBL
)00i62) With respect to antibody-mediated detection and sorting of placental cells, e.g., placental stem cells or placental multipotent cells, any antibody, specific tor a particular marker. can be used, in combination with any fluorophore or either label stiitabtefor the detection and sorting Of cellSje.g., fluorescence-activated cell sorting). Autibody/titiorophore combinations to specific markers include, hut are not limited to, fluorescein isothiocyanate (FlTC) conjugated monoclonal antibodies agaihst HLA<i (availabk fiom Serotec, Raleigh, North Carolina), GDI 0 (available torn BD Immanocytometry Systems, San Jose, California), CD44 (available tom BD Biosciences Pbarmiogett, Sati Jose, California), and CDiOS (available ftorn R&D Systems Inc., Minneapolis, Minnesota); phycoerythrcn (PE) conjugated monoclonal antibodies against CD44, CD200, CDI 17, and CD 13 (BD Biosciences Phamtihgen); phycO«ythrin-Cy7 (PE Gy7) conjugated monoclonal antibodies against CD33 and CDi O (BD Biosciences Pharmingen); allophycocyanin (APC) conjugated streptavidm and monoclonal antibodies against CD38 (BD Biosciences Pharmingen); and Biotinylated CD90 (BD Biosciences Pharmingen). Other antibodies that Can be used include, bttt are W limited to, CD133- AFC (Mihenyi), KDR-Siotin (CD309, Abeam), CytokeratinK-Fitc (Sigma or Dakti), HLA ABC-Fitc (BD), HLA DR,DQ,DP- PE (BD), β-2-microgIobulin-PE (BD)* CD80-PE (BD) and CD86-APC (BD). Other antibody/ label combinations that can be used indude, but are not limited to, CD4$-PerCP (peridin chlorophyll protein); CD44-PE; CD 19- PE; CD tO-F (fluorescein); HLA-G-F and 7- amiuo-aetjnomycinfD (7-AADX HEA-ABOF; and the like· This bat is not exhaustive, and other antibodies from Other suppliers are also commercially available.
1601631 The isolated placental celts provided herein can be assayed for CDI 17 or CD133
Using, forexampfe, phycoerytitrin-Cy5 (PE €y$) conjugated streptavidin and biotin conjugated monoclonal antibodies against CDI 17 or CDI 33; however. Using this system, the cells can appear to be positive for GDI 17 or <pD133e respectively , because of a relatively high background.
{06164} The isolated placental cells cati be labeled with an antibody to a Single marker add defected anti/sotted. Placental cells can also be simultaneously labeled with multiple antibodies to different markers.
{601651 In another embodiment, magnetic beads can be used to stearate cells. The cells may be sorted using a magnetic activated cell sorting (MACS) technique, a method for separating partieles based on their ability to bind magnetic beads (05* lOd pm diameter). A variety of useful modifications can be performed on the magnetic microspheres, including covalent addition of antibody that specifically recognizesa particular call surface molecule or hapten. The beads are then mixed with the cells to allow binding. Cells are then passed through a magnetic field to separate out cells having the specific cell surface marker, ½ erne embodiment, these cells can then isolated and remtixed with magnetic beads coupled to an antibody against additional cell surface markers. The cells are again passed through a magnetic field, isolating cells that bound both the antibodies. Such cells can then be diluted into separate dishes, such as microtker dishes for clonal isolation.
100166) Isolated placental cells can also be charactenzed and/Or sorted based on cell morphology and growth characteristics. For example, isolated placental cells can be characterized as having, and/or selected on the basis of, e.g,, a fibroblastoid appearance in culture. The isolated placental cells can also be characterized as having, ahd/or be selected, cm the basis of their ability to form embryoiddike bodies: in one embodiment, for example, placental cells that are fibroblastoid in shape, express CD73 and CD 105, and produce one or more embryoi<Mike bodies ½ culture are isolated from other placental cells. In another embodiment, OCT-44 placental cells that produce one or more embryotd-like bodies in culture are isolated from etoer placental cells.
100167) hi another embodiment, isolated placental cells can be identified and characterized by a ctiloiiy forming unit assay. Colony forming unit assays are commonly known in the art, such as MesenCult™ medium (Stem Cell Technologies, Inc., Vancouver British Columbia),
(00168) The isolated placental cells can be assessed for viability, proliferation potential* and longevity using standard techniques known in the art, such as trypan blue exclusion assay, fluorescein diacetate uptake assay, pmpidium iodide uptake assay (to assess viability); and thymidine uptake assay, ΜΤΓ (S^^-tiimdhylthiazol-i-ylj-Z^-diphenyUetrazolium bromide) cell proliferation assay (to assess proliferation). Longevity may be determined by methods well know# in toe art, such as by determining toe maximum dumber of population doubling in an extended culture.
|90169{ isolated placental cells, e g., the isolated placental cells described in Sefetroti 4.1, above, cap also be separated from other placental cells using other techniques known ½ toe art, e.g., selective growth of desired Cells (positive selection), selective destruction of unwanted cells (negative seiectio«); sq>aratioh based upondtWential cell aggiutinability in the mixed population as, for example, with soybean agglutinin; freeze-thaw procedures; filtration; conventionaland zonal centrifugation; centritogaUlutriation (counter-streaming centrifugation); unit gravity separation; countercurrent distribution; eIectrophotesis; and the tike.
·:·
4.2.6 Pomilattons of lgolated Placental Cells
100170} Also provided herein am populations of «dated placental cells, e,£;¾ the isolated placental celts described in Section 4. i , above, useful in the methods and compositions described herein. Populations of isolated placental cells can be isolated directly from one or more placenta^ that ¼ the cell population can be a population of placental cells comprising the isolated placental cells, wherein the isolated placental cells are obtained from, or contained within, perfusate, or obtained horn, or contained wititith disrupted placental tissue, e.g< , placental tissue digestate (that; is, the collection of ceils obtained by enzymatic digestion Of a placenta or part thereof). The isolated placental cells described herein can also be cultured and expanded to produce populatkmsof the isolated placental cells. Populations of placental cells comprising the isolated placental cells can also be cultured and expanded to produce placental cell populations.
(00171} Placental Cell populations useful in the methods of treatment provided herein comprise the isolated placental cells, for example, the isolated placental cells as described in Section 4.1 herein, to various embodiments, at least 10%, 20%, 30%, 40%, $ø%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells in a placental cell population are the isolated placental cells. That is, a population of the isolated placental cells can comprise, e,g., as much as 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, We, 90% cells that are not the isolated placental cells.
|0O172| Isolated placental cell populations useful in the methods ami compositions described herein cad be produced by, e.g., selecting isolated placental ceB, whether derived from enzymatic digestion or perfusion, drat express particular markers and/br particular culture or morphological characteristics. In one embodiment for example, provided herein is a method of producing a cell population by selecting placental cells that (a) adhere tb a substrate, and (b) express CD200 and lack expression of HLA-G; and isolating said cells from other cells to form a cell population, to another embodiment, a cell population « produced by selecting placental ceils that express CD200 and lack expression of HLA-O, and isolating said cells from other cells to form a cell population, to another embodiment, a cell population is produced by selecting placental eeltsfoat (a) adhere to a substrate, and (b) express CP73, Cpioi, and Ci>2<N¾ and isolating said cells from other cells to form a cell population, to another embodiment, a cell population is produced by jderrtifyihg placental cells that express CD73, CD 105, and CD200, end isolating said cells from other cells to form a celt population, to Mother embodiment a cell population is produced by selecting placental cells that (a) adhere to a substrate and (b) express (3)200 add OC7V4; teto isolating said cells from other cells to fotin a celt population. In another embodiment, a cell population is produced try selecting placental cells that express CD200 and OCT-4. and isolating said celts from other cells to form a cell population. to another embodiment, a cell population is prodded by selecting placental cells that (a) adhere to a substrate, (t>) express CD73 and CD 105; and (C) facilitate the formation of One or more emfrfyoid-like bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for toe formation of an erobtyOid-like body; and isolating said ceils from other cells to form a cell population, to another embodiment, a cell population is produced by selecting placental cells that express CD73 and CD 105, and facilitate the formation of one or more embtyoid-Iike bodies in a population of placental cells comprising said stem cell when said population is culttoed under conditions that allow for the formation of an embiyoid-iike body , and isolating said celkfirom other cells to form a sell population. to another embodiment, a cell population is prodneed by selecting placental ceils that (a) adhere to a substrate, and (b) express CD73 and CD 105, and lack expression of HIA~G; apd isolating said cells from other cells to fa a cell population; In another embodiment, a cell population is produced by selecting placental cells teat express CD73 and CD105 and lack expression of HLA-G, and isolating said cells from other celb to form a celt population, to mother embodiment, foe method of producing a cell population comprises selecting placental cells that (a) adhere to a substrate, (b) express OCT-4, and (c) facilitate foe formation of one or more emtoyoid-likc bodies in a population of placental cells comprising said stem cell when said population is cultured under conditions that allow for foe formation of an embryoid-like body; and isolating said cells from other cells to form a cell population. In another embodiment, a cell population is produced by selecting placental cells that express OCT-4, and facilitate the formation of one or more embryoid-tike bodies in a population Of placental cells comprising said stem cell when said population is cultured under conditions that allow for the formation of an embryoid-like body, and isolating said cells from other cells to form a cell population. {901T3] to another embodiment, a celtpopuMon is produced by selecting placental cells that (a) adhere to a substrate, and (h) express CD 10 and CD 105, an d do notexpress CD34; and isolating said cells froth other cells to form a cell population In another embodiment, a cell population i$ produced by selecting placental cells that express CD10 and CD 105, and do not express CD34. and isolating said cells Worn other cells to form a cell population, to another embodiment, a cell population is produced by selecting placental cells that (a) adhere to a substrate, and (b) express CD10, CD105, and CD200, and do not express CD34; and isolating said cells from otoer cells to form a cell population, In another embodiment, a cell population ½ prodticed by selectingplacental cells that express CD10, CD105, and CD200, and do w express CD34, and isolating said cells from other cells to form a cell population. In another specific embodiment, a cell population is produced by selecting placental cells that (a) adhere to a substrate, and (b| express CD10, CD90, CD 105 and CD200, and do not express CD34 and CD45; and isolating said cells from other cells to form a cell population. In another specific embodiment, a cell population is produced by selecting placental cells that express CD10, CD90, CDI05 and CO2O0, and do not express CD34 and CD45, and isolating said cells from other cells to form a cell peculation.
{00174] Selection of cell populations comprising placental cells having any of the market combinations described in Section 4.1, above, can be isolated or obtained in similar fashion.
(00175] to any oftiie above embodiments, selection of the isolated cell populations can additionally comprise selecting placental cells that express AB<½ (a placenta-specific ABC transporter protein; see, e.g., Ailikmets ef al., Cancer Res.58(23):5337-9 (1998)), the method can also comprise selecting cells exhibiting at least one characteristic specific to, e,g.? a mesenchymal stem cell, for example, expression of CD44, expression of CD90, or expression of a combination of the foregoing.
{99176] to the above embodiments, the substrate cab be any surihee on which culture and/or selection Of cells, e g., isolated ptaCentot cells, can be accomplished. Typically, the substrate is plastic, e.g., tissue culture dish or multiwell plate plastic. Tissue culture plastic can be coated with a biOmolecule, e.g., laminin or fibifoeectto.
{99177] Cells, e.g., isolated placental cells, pan be selected for a placental cell population by any means known to the art of cell selection. For example, cells can be selected using an antibody or antibodies to one of more cell surface markers, for example, in flow cytometry o* FACS. Selection can be accomplished using antibodies in conjunction with magnetic -beads. Antibodies that are specific for certain stem cell-related markets ate known in the art Fry example, antibodies to OCT-4 (Abeam, Cambridge, MA), 00200 (Abeam), HLA-G (Abeam), CD73 (BD Biosciences Pharmingen, San Diego, CA>, CD105 (Abeam; Bioftesign international, Bacp, MS), etc. Antibodies to other markers are also available commercially, e.g., 0P34, CD38 and CD4S are available from, e.g., StemCell Technologies or BioDesigo International.
(001781 The isolated placental cell populations can comprise placental cells that ate not stem cells, or cells that are not placental cells.
(001791 The isolated cell populations comprising placental derived adherent cells described herein can comprise a second cell type, e,g., placental cells that are not placental derived adherent cells, or, e.g., cells that arc not placental cells. For example, an isolated population of placental derived adherent cells can comprise, e.g., can be combined with, a population df a second type of cells, wherein said second type of Ceil are, e.g., embryonic stem cells, blood cells (e.g., placental blOod, placental blood cells, umbilical cord blood, umbilical cord blood cells, peripheral blood, peripheral blood cells, nucleated cells from placental blood, umbilical cord blood, or peripheral blood, and die like), stem celts isolated Shorn blood (e.g., stem celts isolated from placental blood, umbilical cord blood or peripheral blood), nucleated cells from placental perfusate, e.g., total nucleated cells from placental perfusate; umbilical cord stem cells, populations of blood-derived nucleated cells, bone marrow-derived mesenchymal stromal cells, boue marrow-derived mesenchymal stem cells, bone marrow-derived hematopoietic stem cells, erode tkme marrow, adult (Somatic) stem cells, populations of Stem cells contained within tissue, cultured cells, e.g., cultured stem cells, populations of futiy-diEerentiated cells (e g., chondrocytes, fibroblasts, attimOtic cells, osteoblasts, muscle cells, cardiac cells, etc.), pericytes, and the like. In a specific embodiment, a population of cells comprising placental derived adherent cells comprises placental stem cells or stem cells from umbilical cord. In certain embodiments in which the second type of cell is blood or blood cells, erythrocytes have been removed from the population of cells.
(091S0) In a specific embodiment, the second type of cell is a hematopoietic stem celt
Such hematopoietic stem cells can be, for example* contented within unprocessed placental. ttmbUicalcord blood or peripheral blood; to total nucleated cells from plftoestitol blood, umbilical cord bipod or peripheral blood; m an isolated population of CD344· cells from placental blood, Umbilical potd blood or peripheral blood; to unprocessed tome marrow; to total nucleated cells from bone marrow; to an isolated population ofCD34* cells from bone marrow, or die like,
(60181} In another embodiment, an isolated population of placental derived adherent cells is combined with a plurality of adult otptogehitoi cells from the vaseular system to various embodiments, the cells are endothelial cells, endothelial progenitor cells, myocytes, cardiomyocytes, pericytes, angidblasts, myoblasts Or cardiomyoblasts.
{00182} in a another embodiment, the second cell type is a non-embryonic cell type manipulated in culture in order m express markets Of pltir ipoteneyand functions associated with embiyonicstem cells
160183} In specific embodiments of the above isolated populations of placenta! derived adherent cells, either or both of the placental derived adherent cells and cells of a second type are autologous, or am allogeneic, to an intended recipient of the cells.
{60184} In another specific embodiment, the composition comprises placental derived adherent cells, and embryonic stem cells, to another specific embodiment, the composition comprises placental derived adherent celts and mesenchymal stromal or stem cells, e,g., bone marrow-derived mesenchymal stromal or stem cells. In another specific embodiment, the composition comprises bone matron-derived hematopoietic stem cells. In another specific embodiment, the composition comprises placenta! derived adherent cells ami hematopoietic progenitor cells, e.g., hematopoietic progenitor cells from tome marrow, total blood, umbilical cord blood, placental blood, and/or peripheral blood, to another specific embodiment, tire Composition comprises placental derived adherent cells and somatic stem cells, to a more specific embodiment, said somatic stem cell ¼ a neural stem cell, a hepatic stem celt, a pancreatic stem cell, an endothelial stem cell, a cardiac stem cell, or a muscle stem cell.
{66185} to Other spec ific embodiments, the second type of cells compri se about, at toast, or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of cells in said population. In other specific erobpctoheirts, the PDAC in Said composition comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of cells in said composition, to other specific embodiments, (fie placental derived adherent cells ccanpiise about, at least, or no more than, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 45% ofeel!s in said population.
(60186) Cells in an isolated population Of placental derived adherent cells can be combined with a plurality of cells of another type, e,g>, with a populatibn of stem cells, in a ratio of about 100, 000,000: 1 , 50,000,000: 1, 20,000,000: 1 , 10,000,000:1, 5,000,000:1, 2,000,000:1, 1,000,000:1, 500,000:1, 200,000:1, 100,000:1, 50,000:1, 20,000:1, 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1,5:1,2:1, 1:1: 1:2; 1:5; 1:10; 1:100; 1:200: 1:500; 1:1,000; 1:2,000; 1:5,000; 1:10,000; 1:20,000; 1:50,000; 1:100,000; 1:500,000; 1:1,000,000; 1:2,000,000; 1:5,000,000; 1:10,000,000; 1:20,000,000; 1 :50, 000,000; or about 1:100,000,000, comparing numbers of total nucleated cells in each population. Cells in an isolated population of placental derived adherent cells can be combined with a plurality of cells of a plurality of cell types, as well.
1661871 In other embodiments, a population Of the placental ceils described herein, e.g., the PDACs described above, are combined with osteogenic placental adherent celts (OPACs), eg, the OPACs described in Patent Application No, 12/546, 5S6, ftied August 24, 2009, entitled “Methods and Compositions for Treatment Of Bone Defects With Placental Stem Ceils,” dr combined widi amraon-derived angiogenic cells (AMDACsX the AMDACs described in
U.S. Patent Application No.. 12/622,352, entitled “Amnion Derived Angiogenic Cells”, the disclosure of which is hereby incorporated by reference in its entirety,
43 COMPOSITIONS COMPRISING ISOLATED PLACENTAL CELLS
166188) The placental cells described herein, e.g., in Section 4.1 , can be combined with any physiologicaily-acceptable or ntedically-acceptable compound, composition Or device tor use in tite methods and compositions described herein. Compositions useful in toe methods of treatment provided herein can comprise any one or more of die placental ceils described herein. In certain embodiments, die composition is a pharmaceuticafly-acceptable composition, e>g., a Composition comprising placental cells in a pharmaceutically-acceptable carrier.
!n certain Embodiments, a Composition comprising tiie isolated placental cells additionally comprises a matrix, e.g., a decellularized matrix or a synthetic matrix, to another specific embodiment, said matrix is a three-dimensional scaffold. In another specific embodiment, said matrix comprises collage**, gelatin, lathimti, fibrooectiu, pectin, oroifhme, or vitronectin, in andflier ore specific embodiment, the maw is an amniotic membrane or an amniotic membrmie-derived biomaterial, In .another specific embodiment, said matrix comprises anextiacelhilar membrane protein. In another specific embodiment said matrix comprises a synthetic compound. In another specific embodiment, said matrix comprises a bioactive compound, in atiother specific embodiment said bioactive compound ½ a growth factor, cytokine, antibody, or organic molecule of less than 5,000 daltons.
In another embodiment a composition nsefitl in the methods of treatment provided herein comprises medium conditioned by any of the foregoing placental cells, or any of the foregoing placental cell populations,
4ii
(00191 j The isolated placental cell populations useful m the methods and compositions described herein can be preserved for example, cryopreserved for later use. Methods for cryopreservaiion of cells, such as stem cells, are well known in the art. Isolated placental cell populations cm be prepared in a form that is easily adnunistrable to an individual, e.g., an isolated placental cell population that is contained within a container that is suitable for medical use, Such a container can he, for example, a syringe, sterile plastic bag, flask, jar, or other container from which the isolated placental cell population can be easily dispensed. For example, She container can be a blood bag or other plastic, medically-acceptablebag suitable fbr the intravenous administration ofaliquid to a recipient, the container, in certain embodiments, is one that allows for cryopreseryafibn of the combined cell population.
(00192| The cryopreserved isolated placental cell population can comprise isolated placental cel! derived from a single donor, or from multiple donors; The isolated placental cell population can be completely HLA-matched to an intended recipient, m partially m completely HLArmismatched,
(901931 Thus, ½ one embodiment, isolated placental cells can be used m the methods and described herein in the form of a composition comprising a tissue culture plastic-adherent placental cell population in « Container. In a specific embodiment, the isolated placental cells are cryopreserved. In another specific embodiment, the container is a bag, flask, or jar, Itt another specific embodiment, said bag is a sterile plastic bag; In another specific embodiment* said bag is imiteble for* allows or facilitates intravenous administration pfsaid isolated placental cell population, e.g., by iniravenous infusion. The bag can comprise multiple lumens or compartments that are interconnected to allow mixing of the isolated placental cells and one or more other solutions, e g., a drug, prior to, or during, adramistration. In another specific embodiment, the composition comprises one ortnore compounds that facilitate cryopteservatioft of the combined cell population, th another specific einbodunetit; said isolated placental cell population is contained wttitin a physiologicafly-accep table aqueous solution. In another specific embodiment, said physiolOgically-acceptable aqueous solution is a 0,9% NaCl solution, in another specific embodiment, said isolated placental cell population comprises placental cells that are MLA-matched to a recipient of said cell population. In another specific embodiment, said combined cell population comprises placental cells that are at least partially H LA- mismatched to a recipient of said cell population, in another specific embodimen t, said isolated placental cells are derived from a plurality of donors.
(0Ol94 j Itt cettah embodiments, the isolated placental ceils in the container are isolated
CD 10T, CD34-, CD105+ placental cells, wherein said cells have been crybpreserved, and are contained within a container. In a Specific embodiment, saidCDlOK CD34~, CDlOS-t placental cells are also CD200t. lit another specific embodiment, said CDJO1** CD34-, CD105f , CD2QCH placental cells am also CD4S - pr Ct>9(hh In another specific embodiment, said CDl(H% CD34-, CD 105*, CD20CN- placental cells am also CD45- and CD9tH*. In another specify embodiment die CD34- , CDl(MyCDlO$* placental cells are additionally one or mom of CD 13*, CD29+, CD33+, CD38-, CD44*, CD45-, CD54+, CD62E-, CD62L-, CD62P-, SH3+ (CD73+), SH4* <C&73*>, CD80- CD86-, CD90?, $112* (CD105+), CD106/VCAM*, CDU7-, CD144/VE- cadhertndim, CD184/CXCR4- CD200+ CD 133-, OCT-4+, SSEA3~, SSEA4-, ABC-p+, KDR-<VEOFR2-), HCA-A,B,C*, HLA~DP,DQ,DR- HLA-G-, or Progmmmed Death-1 Ligand (PDLI )+, or any combination thereof, h another specific embodiment, the CD34™, CDlOf, CD105+ placental cells aieadditionally CD 13*, CD29+ CD33+,CD3S~, CD44+, CD45-, CD54/ICAM+ CD62E-, CD62L , CD62P-, SH3*(CD?3*), SH4+ (CD73*), CD80-, CD86-, CD9CH, SH24- (CD1Q5*}, CDl 06/VC AM*, CDl 17- CD14WE-cadherindir¾ CDI84/CXCR4-, CD200f, CD133-, 0CT-4+* SSEA3-, SSEA4-, ABC-jph KDR- (VE0FR2- 4 BLA-A.B.C'h, HLA-<DP,DQ,0R~, HLA'G-, and Programmed Death-1 Ligand (PDLI )t. fflOpSj la certain other embodiments, the above-referenced isolated placenta! cells ait isolated CD200+, HLA-G- placental cells, wherein saidcellshave been cryopreserved, and are contained within «container. In another embodiment, the isolated placental cells are CD73+, CD10S+, CD20ti+ cells that have been cryopreserved^aod are contained within a container. In another embodiment, the isolated placental cells are CD20CH , OCT-4+ stem cells that have been cryopreserved, and are contained Within a container, In another embodiment, the isolated placental cells are CD73+ CD105+ cells that have been cryopreserved, and an? contained within a container, and wherein said isolated placental cells facilitate the formation of one Or more embryoid-like bodies when cultmred with a population of placental cellstrader conditions that allow for the formation of embryoid-tifce bodies, in another embodiment, the isolated placental cells are CD73+, €D105+, HLA-O- cells that have been cryopreserved, and are contained within a container. In another embodiment, the isolated placental cells are OCT-4+ placental cells that have been cryopreserved, and are contained within a container, and wherein said cells facilitate the formation of one or more embtyoid-ltke bodies when cultured with a peculation of placental cells under conditions that allow for the formation ofembryoid-like bodies.
100196) to mtofoer specific embodiment, the above-referenced isolated placental cells are placental stem cells or placenta) multipotenr cells that are CD34-, CD10+ and CD1Q5+ ¼ detected by flow cytometry (e.g., PDACs), In another specific embodiment, foe isolated CD34-i CD 10*, CDI05+ placental cells have the potential to differentiate info cells of a neural phenotype, cells of an osteogenic phenotype, or cells of a chondrogenic phenotype In another specifle embodiment, foe isolated CD34-, GD10+, CD1G5+ placental cells are additionally 002004. Inanofoer specific embodiment, foe isolated C034-, CDJ04, CD105+ placental cells am additionally CD5KH- Or CD45-, as detected by flow cytometiy. In another specific embodiment, foe isolated CD34-, €010+, CD10R placental cells are additionally CD9CH or CD45»:, as detected by flow cytometiy. In another specific embodiment, foe CD34-, CDKH-,
CD 1054, CD200+ placental cells are additionally CD90+ or CD4S-, as detected by flow cytometry, In anofoer specifle embodiment, foe CD34-, CD ICJMvCDlOS*, CD200+ cells are additionally CD90+ and CD4S- as detected by flow cytometry. In another specific embodiment, foe CD34-, CDJ 0+, CD 105* CD29G+, CD904, CD45- bells ate additionally CD80- and CD86-, as detected by flow cytometry; In another specific embddimeot, foe CD34-, CDUH> CDI05+ bells are additionally one ormoie ofCD29+, CD3SK Cp44*, CD54+< Cb$<K CPSfK SH34 or SH4+V In another specific embodiment, the cells are additionally CD44+. In a specific embodiment of any ofthe isolated CE*34~, CD1CH-, CD105+ placental cells above, the celis are additionally one or mote of Cm 17™, 03133-, KDR- (VEGFR2-), HLA-A,B.C+ , flLA^Ol\D&DRn and/or PDLH, f 601971 In a specific embodiment Of any of the foregoing cryopreserved isolated placental cells, said container is a bag. In various specific: embodiments, said container comprises about, at least, or at most i x 10* said isolated placenta! cells, 5x !0* said isolated placental cells, l x to7 said isolated placentalcelfs, 5X 10? said isolated placental cells,lx 10* saidtsolated placental cells, 5 x 10s said isolated placental cells, l x I O9 said isolated placental cells, 5 x 10
: 9 said isolated placenta! cells, 1 x 10** said isolated placental cells, or 1 x 10id said isolated placental celts, toother specific embodiments of any ofthe foregoing cryopreserved populations, said isolated placental celts have been passaged about, at least, or no more than 5 times, no mom than IQ times, no more titan 15 times, or no more than 20 times in another specific embodiment Of any of the foregoing cryopreserved isolated placental cells, said isolated placental celts have been expanded within said container
In certain embodiments, a single unir dose Of placental derived adherent cells can comprise, m various embodiments, about, at least, or no more than 1 x lO3, 3 x 103, 5 x 103, 1 x 104, 3 x 104, 5 x !04, 1 x lO5, 3X lp*, 5 X lO-Yl x 10* 3X 106, 5 x l≠, 1 X W, 3X 10T, 5 x !Q7, 1 x 10s, 3 x 10s, 5 x 10\ I x I09, 5 x lO9, or 1 x 10m placental cells. In certain embodiments, a single unit dose of placental derived adherent celts can comprise between l x IQ3 to 3 x l Q\ 3 x 10* to 5 x IQ3, 5 x 10* to I x iQ4, 1 x 10* to 3 x 104, 3 x l0*to 5 x 104, 5 x 10* to 1 x 10$, I x 105 to 3 x 10s, 3 x 10s to 5X 10*,.5 x IQ$ to 1 x K 1 x 106 to 3 x ίθ6, 3 x 10* to 5 x 106, 5 x lO6 to 1 x IQ7, 1 x l07to 3 x 107, 3 x 107 to 5 x IQ7, 5 x IQ7 to 1 x 10s, 1 x 10s to 3 x 10, 3 x \0 to 5 x 10* $ x 10s to 1 x IQ9 1 x IQ9 to 5 x IQ9, or 5 x 109 to 1 x I Otoplacental Cells. In certain embodiments, the pharmaceutical compositions provided herein comprises populations of placental derived adherent cells, that comprise 50% viable cells or more (that is, at least 50% of the cells in tile population ate functional or living). Preferably, at least 60% of the cells in the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the cells in the population in the pharmaceutical composition are viable.. 43.2
(00198} Populations of isolated placental cells, PDACs, or populations of ceils comprising the isolated placental cells, can be formulated into pharmaceutical compositions for use in vivo, e,g., in the methods of treatment provided herein, Such pharmaceutical compositions comprise a population of isolated placental cells, of a population of cells comprising isolated placental cells, in a pharmaceutically-acceptable carrier^ e.g.f a saline Solution or other accented physiologicatly-aceeptable solution for in vivo administration. Pharmaceutical compositions comprising the isolated placental cells described herein can comprise any, or atty combination, Of the isolated placental cell populations, or isolated placental cells, described elsewhere herein. The pharmaceutical compositions can comprise fetal, maternal, or both fetal and maternal isolated placental cells. The pharmaceutical compositions provided herein can further comprise isolated plhcental cells obtained ½m a single individual or placenta, or from a plurality of individuals or placenme:
(00199} The pharmaceutical compositions provided herein can comprise any number of isolated placental cells. For example, a single unit dose of placental derived adherent cells can comprise about, at least, or no mote titan 1 x IG\ 3 χ 105, 5 x 10*, 1 x 10*, 3 x 104, 5 x 104, 1 x 10s, 3 x 105, 5 x 10s, 1 x 10* 3 x 106, 5 x 10*, 1 x 10?, 3 x 107, 5 x 107, 1 x 10», 3 x 10*, 5 x 10*, 1 X lO9, 5 x 10°, or 1 x 10u) placental cells or between t x 10s to 3 X 10\ 3 x Id3 to 5 X 10\ 5 x I0 j to l x 10*, 1 x 10* to 3 x 10*, 3 x 10* to 5 x 10*, 5 x 10* to 1 x 10* 1 x 10s to 3 x Id5, 3x ll^to 5 X ID5, 5 x 10s to l x 106, 1 X I0*to 3 x 106, 3 x 106to5x 106 5 x 106 to 1 X 107, 1 X I0?to 3 x l07,3 x Id7 to 5 X IO7, 5.x 107 to l x 10*, 1 x 10* to 3 x 10s, 3 x 10s td 5 x 10*, 5 x 10* to I X 10*, 1 x 10¾ tb 5 x 10*, Of 5 x I0¼ 1 x 10lop¼cental cells,
(002001 In certain embodiments, the pharmaceutical compositions provided herein ate administered tb a subject having diabetic peripheral neuropathy once. In Certain embodiments, the pharmaceutical compositions provided herein ate administered to a subject having diabetic peripheral neuropathy bti multiple occasions, e,g., twice, three titties, four times, five times, six times, seven times, eight times, nine times, ten times, or more than ten times. Intervals between dosages can be weekly, bi-weekly, monthly, bi-monthly or yearly. Intervals can also be irregular. Doses ofplaceniat stem ceils administered according to such regimens include, but are not limited to, Ϊ X 107, 3 x 10*, 5 X 103, 1 X IO4, 3 X 10*. 5 x 104, 1 x IO5, 3 X 10*, 5 x 10s, 1 χ 106, 3 X 10* 5 x M* I x !io7, 3 x 10τ > 5 x JO7, ! x 10*, 3 X IO8, 5 X JQ*, I x 109, 5 x or I X 10* placental cells of between I x Id* to 3 x 10*,3 x !0*to $ x 10*, 5 x 10* to 1 x 104, 1 x Id4 to 3 x 10*, 3 x lCr*to 5 x 104, 5 x 104 to J x 10»,.1 x 10s to 3 x 10s, 3 x J0*to5 x 105,5x JO* to 1 x 10®,
1 x 10» to 3 X 10*, 3 x 10* to 5 X 106, 5 x 10* to l x Iti7, 1 x 107 to 3 x lO7, 3 x 107 to 5 x IO7, 5 x 107 to 1 x 108, 1 x 10* to 3 x 10», 3 X 19* to 5 x 10s, 5 x 10* to 1 x 10*, 1 x 10» to 5 x |09, of 5 * lb^to 1 X 10<0 placental stem c6ll$i In a specific embodiment, the dose ofplacental stem cells in aphanuaceutreal composition is I χ 103 placental stem cells, 1» another specific embodiment* the dose Of placental stent cells in a pharmaceutical composition is 3 X 10· placental stem cells.
Id another specific embodiment, ftie dose of placental stem cells in a pharmaceutical composition 'is 3 x W placenta! stem cells» to another specific embodiment, die dose ofplacental stem cells in a pharmaceutical composition is 3 x 10s placental stem cells, to another specific embodiment, the dose ofplacental stem ceils ½ a pharmaceutical composition is 1 x 10® placental stem cells, to another specific embodiment, die dose ofplacental stem cells in a pharmaceutical composition is 3 x 10* placental stem cells. In another specific embodiment, toe dose of placental stem cells in apharmaceutical composition is3 x lO7 placental stem cells.
|O02011 to certain embodiments, a pharmaceutical composition comprising placental Stem cells (e.g,< CD1<K CD105+, cmm, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy once as a single dose. In certain embodiments, a pharmaceutical composition comprising placental stem cells (e.g;, CDS Or, CD105+, CD20Q*, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a Second dose about ! week later, to certain embodiments, a pharmaceutical composition comprising placental stem cells (&.g„ 0>!CH% CD! 05*, CD200*, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about 1 week late- and a third dose about one week after that (i.e., about two weeks after die initial administration). Doses of placental stem cells administered according to such regimens include, bat are not limited to, 1 x IQ3, 3 x 1 ø*, 5 x 103,
1 x 10*, 3 x I04, 5 x 104, ! % 10\ 3 x 10?, 5 x 10?.1 x 10*, 3 x 106, 5 x 106, 1 x Id7, 3 x 107, 5 x 107, .1 x 10s, 3 x to8, 5 x 10s, l x 109, S x lti9, or 1 x 10’* placental ceils or between 1 x jti3 to 3 x Id3, 3 x 103 to 5 x 103, 5 x Id3 to I x I04, 1 x 104 to 3 x 104, 3 x 10*to 5 x Id4, 5 x Id4 to 1 x 105, l x 10» to 3 x 10s, 3 x M5toS x 10s, 5x 10* to 1 x 106, l x 106to3 x 10s, 3x I0*to5x 10*, 5 x 10* to 1 x I07 1 x id7 to 3 x 10?, 3 x 107 to 5x 1 Q7, 5 x to7 to 1 x 10», 1 k 10* to 3 x 10*, 3 X 10* to 5 x 10^5 x 10* to t X 10s, ί x rns to 5 X I0\0if S * 10s to I X lO^piacettial stoto cells. In a specific embodiment, tile dose of placeniaistem cells in a pharmaceutical composition is 1 x 10j placental stem cells, in another specific embodiment, the dose of placental stem cells m a pharmaceutical cotoposition is 3 x 10s placental stem cells, tat another specific embodiment, the dose of placenta! stem cells in a pharmaceutical composition is 3 x ίθ4 placental stem cells. In another specific embodiment, toe dose of placental stem cells in a pharmacetitical composition is 3 x lO5 placental stem cells. In another specific embodiment, the dose of placental stem cells m a pharmaceutical composition is I x 10s placemal stem cells. In another specific embodiment, tiie dose of placental stem cells in a pharmaceutical composition is 3 x 19* placental stem cells.
In another specific embodiment, toe dose ofptacental srem cells m a pbamiacetdic^ composition is 3 x tO7 placental stem cells.
(002021 hr certain embodiments, a pharmacetitical composition comprising placental stem cells (e.g., CDlO*, CD10S^. CDKXH-, CD34- placental stem cells) is administered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about l month later (e.g., about 27, 28, 29, 30> 31, 32, or 33 days after the initial dose). In certain embodiments, a pharmaceutical composition comprising placental stem cells (e.g., CD ICHh, CblOS^ CD200+, CD34- placental stem celts) isadministered to a subject having diabetic peripheral neuropathy as a single dose followed by a second dose about I month later and a third dose about one month after that (i,e„ about two months after toe initial administration, e.g;, on of about day 55, 56, 57, 58, 59, 60, 61, 62, 63, or 64 following the initial administration). Doses of placental stem cells administered according to such regimens include, but are not limited to, 1 x lO3, 3 x 103, 5 k 103, l x Iff, 3 k lO4, 5 x 104, 1 x 19s, 3 x IQ5, 5 k 10*, 1 x 10*.3 x IP6, 5 x 10*, 1 x IQ7 3 X JO7, 5 X tO7, 1 x 10 *, 3 x 10s, 5 x 10s, 1 x IQ9, 5 x 10s, or 1 x 10’* placental cells or between 1 x 103 to 3 x lO1, 3 x 10s to $ x 10* 5 x iO’to 1 x 104, l x 104 to 3 x 104, 3 x 104 to 5 x 10*, -5 x 19* to 1 x 105,
1 x I05 to3 x 10s, 3 x iO*to 5x 10s, 5 x 10s to l x 106, l x 10* to3 x 10s, 3 x 10s to5 x 10s, 5 x 10* to 1 x I07 l x tO7 to· 3 X 107, 3 x lO7 to 5x 1 Q7, 5 x t07to 1 x 10s, 1 x 10s to3x 10s, 3 x tO8 to 5 x 10s, 5 x 10s to 1 x IQ9, 1 x 10s to 5 x 10s, or 5 x 109 to I x 1019 placental stem cells. In a specific embodiment toe dose of placental stem cells in a pharmaceutical composition is l x 193 placental stem dells. In another specific embodiment, the dose of placental stem Cells in a pharmaceutical composition is 3 x 103 placental stem cells. In another specific embodiment, the dote of placental stem cells in a pharmaceutical composition is 3x 19* placental stem cells. In anojrher specific embodiment, the dose of placental stem cells in «pharmaceutical composition is 3 x lO5 placental stem cdljs. In another specific embodiment, the dose ofplacental stem cells in a pharmaceutical composition is 1 K lO6 placental stem cells, in another specific embodiment, the dose Of placental stem cells in a pharmaceutical composition is 3 X, 1 Of placental stem cells. In another specific embodiment, the dose of placental stem cells in a pjmrmaceutical composition is 3 x i07 placental stem celts.
(60203) The pharmaceutical compositions provided herein Comprise populations of cells that comprise 50% viable cells or titoire (that is, at least 50% Of the cel ls in the population am functional dr living). Preferably, at least 60% of tire cells m the population are viable. More preferably, at least 70%, 80%, 90%, 95%, or 99% of the ceils in the population ip die pharmaceutical composition are viable;
(602041 The pharmaceutical compositions provided herein can comprise one Or more compounds that* «,$.* facilitate engrafiment (e.g., anti^T-cCll receptor antibodies, an immunostippressant, or die like); stabilizers such as albumin, dextran 40, gelatin, hydroxyethyl starch, plasmalyte, and the like.
(662651 When formulated as «8 injectable solution, in one embodiment the pharmaceutical composition comprises about 1% to 1.5% ESA and about 2,5% dextran. In a preferred embodiment, the pharmaceutical «imposition comprises from about § x 106 cells per milliliter to about 2 x 10* cells per milliliter m a solution comprising 5 % HSA and 10% dextran, optionally comprising an immunosuppressant, e.g., cyclosporine A at, e.g., 10 ntg¾.
(002061 In other embodiments, the pharmaceutical composition, e.g., «solution, comprises a plurality of cells, e.g., isolated placental cells, for example, placental stem cells or placental multipotent cells, wherein said pharmaceutical composition comprises between about L0 *0.3 x
TO6 cells per milliliter to about 5.0 ± ! .5 x I06 celh per milliliter. In other embodiments, the pharmaceutical composition comprises between about 1.5 x 106 celts per milliliter to about 3.75 x 10* cells per milliliter. In Other embodiments, the pharmaceutical composition comprises between about 1 x !0* cells/mL to about 50 x 10s ce!is/mL, about 1 x l06 cells/mL to about 40 x 10* cells/mL, about I x 10* cells/mL to about 30 x 10* cells/mL, about ) x 10* celfs/mL to about 20 x 10* cells/mL, about I x !O6 cetis/mL to about 15 x 10s cells/mL, or about 1 X JO* cells/mL to about 10x 10* cells/mt . In certain embodimen ts, the pharmaceutical composition comprises ho visible ©ell clomps (U., no tiraerocell clumps), or substantially nosuch visible clumps,: As used hereby ‘"macro cbticlbmps^means an aggregation of cells visible without magnification, e.g., visible to the naked eye, and generally refers to a cell aggregation larger than about 150 microns In some embodiments, the pharmaceutical composition comprises about 2.5%, 3.0%, 3.5%, 4,0%, 4.5%, 5.0%, 5.5%, 60%, 6.5%, 7.0%, 7.5% 8,0%, 8.5%, 9,0%, 9.5% or 10% dextran, e:g., dextran-40, in a specific embodiment, said composition comprises about 7>5% to about 9% dextran-40. In a spec ific embodiment, said composition comprise about 5-5 % dextrrm-40, in certain embodiments, the pharmaceutical composition comprises from about 1% to abemt 15% human seniUi albumin (HSA). In specific embodiments, the pharmaceutical composition comprises about 1%, 2%, 3%, 4%, 5%, 65, 75, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% HSA. In a specific embodiment, said cells have been cryopreserved and thawed. In another specific embodiment, said Cells have been filtered through a 70 pM to 100 μΜ filter, in another specific embodiment, said composition comprises no visible ceil clumps, in another specific embodiment* said composition comprises fewer than about 200 ceil clomps per 106 cells, wherein said cell clumps are visible only under a mierpseope, e.g., a, tight microscope. In another specific embodiment, said composition comprises fewer than about 1 $0 cell clumps per 10* cells, wherein said cell clumps are visible only under a microscope, e.g., a tight mibrbscope.. Γη another specific embodiment, said composition comprises fewer than about 100 celt clamps per 10* celts, wherein said cell clumps ate visible only under a microscope, e.g., a light microscope.
(002071 In a spetific embodiment, the pharmaceutical composition comprises about 1.0
*0.3 x 10* cells pen milliliter, about 5.5% dextran-40 (w/v) , about 10% HSA (w/y), and about 5% DMSO (v/v). In another specific embodiment, a pbarmaceuttcal composition comprising placental stem cells provided herein comprises about 5.75% dextrin 40, about 10% human serum albumin, and about 2.5% DMSO.
(00208 j In other embodiments, the pharmaceutical composition comprises a plurality of qe¾ e,g„ a plurality of Isolated placental cells in a solution comprising 10% dextran-40, wherein fee pharmaceutical composition comprises between about 1.0 ± 0.3 x 10* cells per milliliter to about 5.0 ,*1.5 x 10* cells per milliliter, and wherein said composition comprises so cell clumps visible with the unaided eye (i.e., comprises no macro cell clumps). In some embodiments, the pharmaceutical composition comprises between abbot 15 x 10* cells per milliliter to about 3,75 x 10* cells per milliliter, to a specific embodiment said cells have been cryopreserved and thawed. In another specific embodiment, said cells have been filtered through a 7QpM to 100 μΜ filter. In another specific embodiment, said composition comprises fewer than about 200 micro cell clumps (that is, cell clumps visible only with magnification) per 10* cells. In anothetspecific embodiment, me pharmaceutical composition comprises fewer than about 150 micro cell chimps per 1# cells. tn another specific embodiment, the pharmaceutical
: composition comprises fewer titan about 100 micro cell dumps per JO* cells, to another specific embodiment the pharmaceutical composition comprises less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%,: 4%, 3%, or 2% DMSO, or less than 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0,3%, 0,2%, or 0.1% DM$0.
1002001 further provided herein are compositions comprising cells, wherein said compositions ate produced by one of the methods disclosed herein. For example, in one embodiment, the pharmaceutical compo&Hion comprises cells, wherein the pliatmaceutical composition is produced by a mtihod comprising filtering a solution comprising placental cells, e,g„ placental stem cells or placental multipoient cells, to form a filtered cell-containing solution; diluting the filleted cell-contaimiig solution with a first solution to about I to 50 x 10s, 1 to 40 x 106, 1 to 30 x 10* t to 20 x 10*, I to 15· x I0*, or 1 to 10 x i0*ed&pe? milliliter* e.g., prior to cryopreservation; and diluting the resulting filtered cell-containing solution with a second solution comprising dextrah, but not comprising humatt serum albumin (USA) to produce said composition. In certain embodiments, said diluting is to no more than about 15 x 10* cells per milliliter. In certain embodiments, said diluting is to no mote than about 10 * 3 x 106 ceils per milliliter. In certain embodiments, said diluting is to no more than about 7,5 x 10* cells per mi lliliter. In other certain embodiments, if the filtered cell-containing solution, prior to the dilution, comprises less titan about 15 x 1 O* cells per milliliter, filtration is optional. In other certain embodiments, if the filtered cell-containing solution, prior to the dilution, comprises less than about 10 * 3 x 19* ceils per milliliter. filtration is optional. In ptiier certain embodiments, if the filtered cell-containing solution, prior to the dilution, comprises less than about 7.5 x 10s cells per milliliter, filtration is optional
1002101 Iti a specific embodiment, the cells are ciyopreserved between said diintmg with a first dilution solution and said diluting with said second dilution solution. In another specific embodiment, the first dilution solution comprises dextran mad HSA. The dextran in the first dilution solution or second dilutiou solution can be dextrah of any molecular weight, e,g», d&ttran having a molecular weight of from about 1 O kDa to about 150 kDa. to some embodiments, said dextran in said first dilution solution or said second solution is about 15%, 3.0%, 3,5%, 4,0%, 4.5%, 5.0%, 53%, 6,0%.6.5%, 7,0%, 7.5% 80%, 8,5%,: 9,0%, 9.5% or 10% dextran. to another specific embodiment, toe dextran in said first dilution solution or said second dilution solution is dextran-40. to attother specific embodiment, toe dextran in said first dilution solution mid said second dilution solution is dextian-40, to another specific embodiment, said dextran-40 in said first dilution solution is 5.0% dextran-40. In another specific embodiment, said dextran-40 in said first dilution solution is 5.5% dextran-40; In another specific embodiment» said dextran-40 in said second dilution solution is 10% dextrau-40. to another specific embodiment, said HSA in send solution comprising HSA is 1 to 15 % HSA. In another specific embodiment, said HSA in mid solution comprising HSA is about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10¼, 11%, 12%, 13%, 14% or 15 % HSA. to another specific embodiment, said HSA to said Solution comprising HSA is 19% HSA. In another specific embodiment, said First dilution solution comprises HSA- to another specific embodiment, said HSA in said first dilution solution is 10% HSA. to another specific embodiment, said first dilution solution comprises a ccyoprotectant. to another specific embodiment, said ctyopratectant is DM$Q* to another specific embodiment, said dextran-40 in said second dilution solution is about 10% dextran-40. In another specific embodiment, said composition comprising cells comprises about 7,5% to about 9% dextran. to another Specific embodiment, the pharmaceutical composition comprises from about 1.0 *0.3 x 1 (C ecils per milliliter to about 5.0 *1.5 x 10* cells per milliliter. In another specific embodiment, the pharmaceutical composition comprises from about 1,5 x l<f cells per milliliter to about 3.75 x W celts per milliliter. to another embodiment, toe pharmaceutical composition is made by a method comprising (a) filtering a cell-containing solution comprising placental cells, e.g., placental stem cells or placental multipotent cells, prior to cryopreservation to produce s filtered cell-containing solution; (b) cryopreservmg the cells in the filtered cell-containing solution at about 1 to 50 x 10», 1 to 40 x If, 1 to 30 x W, I to 20 x W, 1 to 15 x 10*, or 1 to 10 x W cells per milliliter; (c) thawing the cells; and (d) diluting toe filtered cell-containing solution about 1 il to about 1 ill
(v/v) with a dextran-40 solution, to certain embodiments, if the number of cells is less titan about 10 i 3 x 10s cells per milliliter prior to step (a), filtration is optional, to another specific embodiment» the cells in step (b) are ctyopreserved at about to .·£·.3 x lO6 cells per totHititeir. til another specific embodiment, the cells in step (b> are cryopreserved in a solution comprising about 5% to about ip% dextran-40 and MBA. in certamembodiments, said diluting in step (b) is to no more tban about 15 x .10* cells per milliliter.
(00212| In another embodiment, the pharmaceutical composition is made by a method comprising', (a) suspending placental cells, placental stem coils or placental retiltipotent cells, in a 5.5% dextran-40 solution that comprises 10% HSA to form a cell-containing solution; (b) filtering the cell-containing solution through a 70 pM filter, (c) dilating the cell-containing solution with a solution comprising 5.5% dextran-40, 10% HSA, and 5% DMSO to about 1 to 50 X 106, 1 to 40 x 10s, I to30x 106, 1 to20x 10s, 1 to J5x t06,or 1 td lOx 10* cells per milliliter, (d) cryopreservtng the cells; (e) thawing to* cells; and (f) diluting the cell-containing solution 1 : 1 to 1 : 11 (v/v) with 10% dextran-40. In certain embodiments, said diluting to step(c) is to no more tban about 15 x 106 cells per milliliter. In certain embodiments, said diluting in step (c) is to no more than about 10 *3 x 10* cells/mL. In certain embodiments, said diluting in step (c) is to no mote than about 7.5 x 10* cells/mL.
(00213] In another embodiment, the compositiitto comprising cells is made by a method comprising: (a) centrifuging a plurality of cells to collect the cells; (b) resuspending the cells to 5, 5% dexti¾»«40; (c) cenuifuging the cells to collect toe cells; (d) resuspending toe cells in a 5,5% dextran-40 solution Aat comprises 10% HSA; (e) filtering the cells through a 70 μΜ filter, (i) diluting the cells in 5:5% dextran-40, 10% HSA, and 5% DMSO to about 1 to 50 * lot I to 4OX 106, 1 to 30x 10*, 1 toTOx IQ6, I to l5.x ld6, or 1 to 10 x 10* cells per milliliter; <g) cryopresetvmg the cells; (hj thawing the cells; and (i) diluting toe cells 1 :1 to 1 :1 i (v/v) with 10% dextran-40. In ccmto anbodtments, said diluting to step (f) is to no more than about 15 x lO6 cells pertoftlititer. In certain embodiments, said diluting in step (f) is to no more than about 10 ±-3 x l O6 cells/mL. In certain embodiments, said diluting in step (f) is to no more than about 7.5 x 10* Cells/mL. In other certain embodiments, if toe number of cells is less than about 10 db 3 x 10* celts per miflilhet, filtration is optional.
(00214] The compositions, e.g., pharmaceutical compositions comprising the isolated piacental cells. described herein can comprise any of the isolated placental cells described herein. t¾i02!5I Other injectable formulations, suitable tor the administration of cellular produces, maybeused.
(00216) In one embodiment, the pharmaceutical composition comprises isolated placental cells that are substantially, orconrpletely* tton-materaal in origin, that is, have the fetal genotype; e.g., at least about 90%, 95%, 98%, 99% or about 100% are non-matemal in origin. For exatnple¾½ one embodiment a pharmaceutical composition comprises » population of isolated placental cells that ate CD200+ and HLA-G-; CD73+, CDIG5+, and CD20&+; CD2(KH· and OCT-4+; CD73+, CD ! 05+ and HLA-G-; CD73+ andCDl05+ and facilitate the formation of one or more embiyokMike bodies in a population of placental cells comprising said population of isolated placenta! cell when said population of placental celts is cultured under conditions that allow the formation of an embryoid-iike body ; or OCI>4+ and facilitate the formation of one or more embfyoid-likebodies in a palliation of placental cells comprising said population of isolated placental cell when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like body; or a combination Of the foregoing, Wherein at least 70%, 80%, 90%, 95% or 99% of said isolated placental cells are non-maternalih origin. Jin another embodiinetit, a pharmaceutical composition comprises a population of isolated placental cells that am CDitK CD1GS+ and CD34-; CDl<H , CD 105+, 00200+ and CD34-; CDltK CDl05t, CD200+, CD34 " and at least one of CD90+ or CD45-; CDKH, CD90+, CD105+, CD20Q+, CD34- andCD45~; CD10+, CD90+, CD1G5+, CD200+,€D34- and CD45-; CD20(H and HLA-G-; CD73+, CD105+ and CD200+; CD2004· and OCMn CD73+, CD1054· and HLAdS-s CD73+ and CD 105+ and facilitate the formation Of One or more embryoid-like bodies m a popplfttion of placental cells comprising said isolated placental cells when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like body; OCT-4+ and facilitate the formation of one or mote embryoid-like bodies in a population of placental cdls comprising said isolated placental cells when said population of placental cells is cultured under conditions that allow the formation of an embryoid-like body; or one or more of CD 117-, ^D13> , KDR VCD80-, CD86-, ΗίΑ-Α,Β,Ο+, riLA~DP,DQ,DR afid/or PDLI+; or a combination of the foregoing, wherein at least 70%, 80%, 90%, 95% or 99% of said isolated placental cells are non-maternal in origin. In a specific embodiment, the pharmaceutical composition additionally comprises a stem cell that is not obtained from a placenta. (802171 Isolated placental calls in the compositions, e g., pharmaceutical compositions, provided herein, can comprise placental cells derived from a single donor, or from multiple douora Hie isolated placental cells can be completely HL A-matched fo an mlerided tecipient, or partially ojrcompletely HLAmmmatched.
03 Matrices Comprising isolated Placental Cells
(802181 Further provided herein am compositions comprising matrices, hydrogels, scaffolds, and the like that comprise a placental cell, m a population of isolatedplacental cells. Such compositions can he used in the place Of, or in addition to, cells in liquid suspension.
1002191 The isolated placental ceils described heretocan be seeded onto a natural matrix, e.g,, a placentalbiomaterial such as an amniotic membrane material. Such an amntotic membrane material can be, e.g., amniotic membrane dissected directly from a mammalian placenta; fixed or heat-treated amniotic membrane, substantially dry (i.e., <20% H20) amniotic membrane, chorionic membrane, substantially dry chorionic membrane, substantially dry amniotic and chorionic membrane, and the like. Preferred placental biomatetials cm which isolated placental cejls can be seeded are described in Hariri, U.S. Application Publication No. 2004/0048796, the disclosure of which is incorporated herein by reference to ½ entirety ,
([002201 The isolated placental cells described herein can be suspended in a hydrogel solution suitable for. e.g., injection; Suitable hydrogels for such compositions include self- assembling peptides, such as RAD 16. In rate embodiment, a hydrogel solution comprising the cells can be allowed to harden, for instance in a mold, to form a matrix having cells dispersed therein for implantation. Isolated placental cells to such a matrix can also be cultured so that the cells are mjtotically expanded prior to implantation. The hydrogel is. e.g„ an organic polymer (natural or synthetic) that is cross-linked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure that entraps water molecules to form a gel. Hydrogel-forming nwterials include polysaccharides such as alginate and salts thereof peptides, poiyphosphaztnes, and polyacrylates, which toe crossimked ioriically, or block polymers such is polyethylene oxide-polypropylene glycol block copolymers which are crosslinkedby temperature or pH, respectively, in some embodiments, the hydrogel or matrix is biodegradable.
(80221} In some embodiments, die formulation comprises an in situ polymerisable gel (see,> e.g., U S. Patent Application Publication 2002/0022676, toe disclosure of which is incorporated herein by reference in its entirety; Ansefe et al„l Control Release, 78(1-3): 199- 209 (2002); Wang et al., Biomaterials, 24<22):3969-80 (2003).
(00222} In some embodiments, the polymersare at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions, that have charged side groups, or a monovalent ionic salt thereof. Examples of polymers having acidic side groups that can be reacted With cations are poiyiphospha^nes), p«ty(acfyiic acidaX poly(mefoacryHc acids), copolymers of acrylic acid and methacrylic acid, polyfvinyl acetate), andsulfonated polymers, such as sulfonated polystyrene. Copolymers having acidic side groups formed by reaction of acrylic or methacrylic acid and vinyl ether monomers or polymers can also be used. Examples of acidic groups arc carboxylic add groups, sulfonic add groups, halogenated (preferably fiuorihated) alcohol groups, phenolic QH groups, and acidic QH groups.
(902231 In a specific embodiment, the matrix is a felt, which can be composed of a multifilament yam made from a bioabsorbable material, e.g., PGA, PLA, PGL copolymers or blends, or hyaluronic acid. The yarn is thade into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling, to another preferred embodiment the cells of the invention am seeded onto foam scaffolds feat aiay be composite structures. In addition, fee three-dimensional framework may he molded into a useful shape, such as a spetific structure in the body to be repaired, replaced, or augmented. Other example of scaffolds feat can be used include nonwoven mate, porous foams, or self assembling peptides. Nonwoven mats can be formed using fibers comprised of a synthetic absorbable copolymer of glycolic and lactic acids (e.g., PGA/PLA) (VlCPYL, Ethicon, toe., Somerville, N J.). foams, composed of. e.g„ poly(e-caprolactone)/poly(glycolic acid) (RCL/PGA) copolymer, formed by processes such as freeze-drying, or lyophilization (see, e.g., O.S. Pat No. 6,355,699), can also be used as scaffolds.
(002241 The isolated placental cells described herein or cd-cultures thereof can be seeded onto a feree-dimensiotial framework or scaffold and implanted in vivo. Such a framewodi can be implanted in combination with any one or more growth fectors, ce¾ drugs or other components feat, e g., stimtilate tissue formation.
(00225| Examples of scaffolds that can be used include nonwoven mats, porous foams, or self assembling peptides. Nonwoven mats can be formed using fibers comprised Of a synthetic absorbable copolymer oigiycOlicand Me acids (e.g., PdA/PLA) (VICRYL, Efoicoh, foe,, Somerville, NIL). Foams, composed of, e<g., poly(e-caprolactoneXpo]y(jgIycolic acid) (PCE/PGA) copolymer, formed by processes such asffeeze-drying, or lyophilizatiou (see, e.g., O.S. Pat. No, 6355,699), can also be Used as scaffolds.
(60226} In another embodiment, isolated placental cells can be seeded onto, or contacted with, a felt, which edit be, e.g., competed of a muttifilameiit yam made froro a btoabsorbable materia! such as JP<3 A, PLA, PCL copolymers or blends, or hyaluronic acid.
(60227| The isolated placental cells provided herein can, in another embodiment, be seeded onto foam scaffolds that may be composite structures. Such foam scaffolds can be molded into a useful shape, such as that of a portion of a specific structure in the body to be repaired, replaced oraugmented, lit some embodiments, the ftameworkis treated, e.g., with 0.1 M acetic acid followed by incubation in polylysine, PBS, and/or collagen, prior to inoculation of foe cells in order toenhatice cell attachment. External surfaces of a matrix may be modified to improve foe attachment or growth of cells and differentiation of tissue, such as by plasma- coating the matrix, or addition of one or more proteins (e.g., collagens, elastic fibers, reticular fibers), glycoproteins, glycosammogiycans fog. , heparin sulfate, chonfooitin*4-stilfate, chondroitin-ti-sulfate, dermatan sulfate, keratin sulfate, etc.), a cellular matrix, and/or other materials Such as. but not limited to; gelatin, alginates, agar, agarose, and plant gums, and foe like.
(60228| in some embodiments, the scaffold comprises, or is treated with, materials that render it noo-fotombogenjc, These treatments and materials may also promote and sustain endothelial growth, migration, and extracellular matrix deposition. Examples of these materials and treatments include but are not limited to natural materials such as basement membrane proteins such as laminin and Type IV collagen, synthetic materials such as EPT.FE, and: segmented polyurefoaneurea silicones, such as PURSPAN™ (The Polymer Technology Group, Inc., Berkeley, The scaffold can also comprise anti-thrombotic agents such as heparin; the scaffolds can also be seated lo alter foe surface charge fog.* coating with plasma) prior to seeding with holated placentai cells,
(062291 The placental cells fog., PDACs) provided herein can also be seeded onto, or contacted with, a physiologically-accq)table ceramic material including, but not limited tb, mono-. dk tri-, elpha-trkbetartri-, atid teira-calcium phosphate, hydroxyapatite, fluoro^tites, calcium sulfates, calcium fluorides, calcium oxides, calcium carbonates, magnesium calcium phosphates, biologically active glasses such as BlOGLASS$, and mixtures thereof. Porous biocompatible eeiamic materials currently commerciallyavaiWle include SURG1BGNES (CsnMedica Coip., Canada), ENDOBON® (Merck Biomaterial France, Fmee), CEROS® (Mathys, AO, Bettlach, Switzerland), and mineralized collagen bone grafting products such as HEALOS™ (DePuy, Inc., Raynham, MA) and V!TOSS®, RHAKQSS™ aiid CORTOSS® (Orthovita, Malvern, Pa). The framework can be a mixture, blend or composite of natural and/or synthetic materials,
1002361 in one embodiment, the isolated placental cells are seeded onto, or contacted with, a suitable scaEold at about 0.5 x IQ* to about 8x 10* eefts/mL
Treatment of Diseases
#602311 Provided herein ate methods of treating an individual having a disease, disorder or condition, wheteiu the disease, disorder or condition is caused by , or ½ associated with, an inappropriate or undesirable immune response, e.g., a disease, disorder or condition that can be treated beneficially by immunosuppression, comprising administering to the individual placental stem cells, to a specific embodiment, the amount is an amount sufficient to delectably suppress an immune response in the individual Such ah immune response can be, e g., proliferation of T cells in an MLR or regression assay performed using T cells from the individual #602321 Ah individual having a disease, disorder or condition associated with or caused by an inappropriate or undesirable immune response, e.g., an individual having, or at risk of developing multiple sclerosis; a person having, or at risk of developing, an inflammatory bowel disease, e g., Crohn's disease or ulcerative colitis; a person having or at risk of developing graft* Vc¾sus-host disease; a petscm having of at risk of developmg scleroderma; a person having or at risk of developing rheumatoid arthritis; a person having or at risk of developing diabetes, a person having or at risk of developing psoriasis; a person having or at risk of developing mycosis ftmgoides; and the tike, can be treated with a plurality of placental stem ceils, and, optionally, one or more therapeutic agents, at any time during the progression of the disease. For example, the individual can be treated immediately after diagnosis, or within 1 , 2, 3, 4, 5, 6 days of diagnosis, or within l, 2, 3, 4, 5, 6, 7, 8, 9, I D, 15, 20, 25, 30, 35, 40, 45, 50 or mote weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years after diagnosis; The individual can be treated puce, or multiple times during the clinical course of die disease. The individual can be treated, as appropriate, during an acute attack, during remission, or during a chronic degenerative phase, f 002331 The placenta stem cel ls usefiil h die treatment of suc h a disease, disorder or
Condition can be any of the placental stem cells disclosed herein. In a specific embodiment, the placental stem cells express CD200 and HLA-G; express CD73, CD105, and CD200, express CD200 and OCT4; express €D73, GD105 and HLA-G; express CD73 and CPIOS, and facilitate the formation of one of more embryoid-like bodies in a population of placental cells when said population ¼ cultured under conditions that allow for rite formation of embryoid- Hke bodies; Or expressCK’TA, and (c) fedlitate the formation of one ortnire embryoid-Bke bodies to a population of placental cells wheat said population is cultured under conditions that allow ¾ the formation of embryoid-like bodies; or any combination of the foregoing. In a specific embodiment, the placental stem cells are CDWs, CD 1054, CD20Q+, CD34*1 placental stem cells. in another specific embodiment, the placental stem cells are CD! 174.
1602341 In one embodiment, the individual is administered » dose Of about 300 million placental stem cells. Dosage, however, can vary according to toe individual's physical characteristics, e.g., weight, add can range from 1 miilion to 10 billion placental stem cells per dose, preferably between 10 million and I billion per dose, or between 100 million and 50 million placental stem cells per dose. The administration is preferably intravenous, but can be by any medicalty-acceptable route for the administration of live cells, e.g., paremerally, subcutaneously, intramuscularly, intraperitoneally, intraocularly, and the like. Id one embodiment, the placental stem cells are from a cell bank. In one embodiment, a dose of placental stem cells, e.g., from amnion, amnion/chorion, chorion or umbilical cord, is contained within a blood bag or similar bag, suitable tor bolus injection or administration by catheter, 1002351 In another embodiment, provided herein is a method of treating an indi vidual having a disease, disorder or condition, wherein the disease, disorder or condiuon is caused by, or is associated with, an inappropriate or undesirable immune response, e.g., a disease, disorder or condition that can be treated beneficially by immunosuppression, comprising administering to the ifldi vidiml euhute medium that baa been conditioned placental stem cells, in an amount sufficient to detectably suppres s an immune response in the indi vidual. Such an immune response can be, eg., proliferation of T dells in an MLR or regression assay performed using T cells from the individual. {88236} Placental stem ceils or umbilical cord stem celts, or medium conditioned by placental stem celUt or umbilical cord stem ce¾ can be administered ½ a single dose, or in multiple doses. Where placental stem cells are administered in multiple doses, the doses can be part of a therapeutic regimen designed to relieve one. or more acute symptoms of disease, disorder or condition, wherein the disease, disorder or condition is caused by* or is associated with, aft inappropriate or undesirable imtntitte response, or can be part df a long-term therapeutic regimen designed to prevent, or lessen the severity, of a chronic course of such a disease, disorder Or condition.
Treatment of Multiple Sclerosis j802371 In another aspect, provided herein is a method of treating an individual haying multiple sclerosis, or a symptom associated with multiple sclerosis, comprising administering to die individual a plurality Of placental stem cells, or medium conditioned by placental stem ce¾ in an amount and for a time sufficient to detectably modulate, e.g., suppress an immune response in the individual. j 80238 j Multiplesclerosis (MS) is a chrome, recurrent inflammatory disease of the central
Uervous sEystem. The disease results in injury to the myelfo sheaths surrounding CNS and PNS axons, oligodendrocytes; and the nerve cells themselves. The disease is mediated by autoreactive T celts, particularly CD4+ T cells, that proliferate, cross foe hloodforain barrier, mid enter die CNS under the influence of cellular adhesion molecules and pro-mflammatoiy cytokines, lire symptoms Of MS include sensory disturbances in the limbs, optic nerve dysfunction, pyramidal tract dysfunction, bladder dysfunction, bowel dysfunction, sexual dysfunction, ataxia, and
|00239J Four different types or clinical courses of MS have beat identified. The first, relapsing/temitting MS (RRMS) «characterized by self-limiting attacks of neurological dysfunction that manifest acutely, over the course of days to weeks, followed by a period of recovery, sometimes incomplete, over several months. The second type, secondary progressive MS (SPMS), begins as RRMS but changes such that the clinical course becomes characterized by a steady deterioration m function unrelated to acute attacks, the third, primary progressive MS (FffMS), is characterized by a steady d.ecline in function from onset, with no acute attacks. The fourth type, progressi ve/relapsing MS (PRMS), also begins with a progressive course, with occasional attacks superimposed on the progressive decline in function. 00240 Person® having MS are generally evaluated usihga motor skii jts assessment, optionallywitb an MRL For example» one motor skills assessment, the expanded disability stams scale, scores gradations h an affected individual's abilities, as follows:
0.0 Normal neurological examination.
1,0 No disability, minimal signs in one FS<
15 No disability, minimal signs id more than ofle FSi 2.0 Minimal disability in one FS.
2.5 Mild disability in One FS dr mmimal dtsability in wo FS.
3-O Moderate disability in one FS, or mild disability in three or four FS. Putiy ambulatoty.
35 FUlly ambulatory but with moderate Usability in one FS and more than minimal disability in several others.
4.0 Fully ambulatory without aid, self-sufficient, up and about some 12 hours a day despite relatively severe disability; able to walk without aid or rest some 500 meters.
45 Fully ambulatory without aid, up and about much of the day, able to Work a full day, may otherwise have some limitation of full activity or require minimal assistance; characterized by relatively severe disability; able to walk without aid or rest some 300 meters. 5.0 Ambulatory without aid or rest for about 200 meters; disability severe enough to impair full daily activities (work a foil day without special provisions) 5.5 Ambulatory without aid or rest for about 100 meters; disability severe enough to preclude foil daily activities 6.0 Intermiitenf or unilateral constant assistance (cane, crutch, brace) required to walk about 100 meters with or without resting 6.5 Constant bilateral assistance (canes, crutches, braces) required to walk about 20 meters without resting.
7,0 Unable to walk beyond approximately five meters even with aid, essentially restricted to wheelchair; wheels self in standard wheelchair and transfers alone; up and about in wheelchair some 12 hours a day.
7.5 Unable to take more than a few steps; restricted to wheelchair; may need aid in transfer; wheels self but cannot carry on in standard wheelchair a foil day; May require motorized Wheelchair
8.0 Essentially restricted to bed or chair or perambulated in Wheelchair, but may he out of bed itself much of the day; retains many selfcare functions; generally has effective use of arms 8.5 Estetitiafly resided to bed touch of day, has some effective rise of arms plains some self care functions.
9,0 Confined to bed; can still commimicate and eat
9,5 Totally helpless bed patient; unable to communicate efiWVbly or eat/swallow 10,0 Death doe to MS,
(00241) Ih die above scoring system» ’Τ¾" teferstb the eight functional systems measured, incltttting pyramida!, ceirebellar, brainstem, sensory, bowel and bladder, v&ualv<terebral, and othersy stems
Other, similar scoring systems are known, including the Scripps neurOlogicai rating scale, the ambulatory index, and the multiple sclerosis iuncti onal composite score ( MS FC). (0232] The progress of M S has also been assessed by a determination of die attach rate. [0233] The progress of MS has also been assessed by magnetic resonance imaging, which cab detect neural lesions associated with MS (e.g., new lesions, enhancing lesions, or combined unique active lesions): [0234] Thus1, in one embedment, provided hereto is a method of treating an individual having MS, e.g., and individual who has been diagnosed with MS, comprising administering to the individual a plurality of placental stem cells sufficient to delectably suppress an immune response ¾ the individual. ¼ a Specific embodiment, the MS is relapStog/itmftting MS. In another specific embodiment, the MS is secondary progressive MS. In another specific einbodiment, the MS is prhnary progressive MS. In anotoer specific embodiment, the MS is progressive/relapsing MS. In another specific embodiment, the administering delectably improves one or more symptoms of MS in the individual, In toOte specific embodiments, the symptom is, e.g., one or more of a sensory disturbance in the limbs, an optic nerve dysfunction, a pyramidal tmctdysfunction, a bladder dysfunction, a bowel dysfunction, a sexual dysfunction, ataxia, or diplopia. In another specific embodiment, saidadministering results in an improvement on the BDSS scale of at least one half point In another specific embodiment, said administering results in the maintenance of function, according to at least one MS scoring system, over the course of, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, In another specific embodiment, said administering results in an improvement on the BOSS scale of at least one point In another spedfic embodiment, said administering results in an improvement on the EDSS scale of at least two points. In other specific embodiments, said administering results in a detectable improvement on a multiple sclerosis assessment scale or bn an MRl The individual can be treated, as appropriate, duringunacute attack, during remission, of during a chronic degenerative phase. to another embodiment, the placental stem cells ate administered to a female having MS* post-partuiu, to maintain the state of remission or reduced occurrence of relapse experienced during pregnancy,
(002431 Also provided herein ate methods for the treatment of an indi vidual having MS, e<g„ an individual who has been diagnosed as having MS, comprising administering to the individuala plurality of placental stem cells sufficient to detectab!y suppress an immune response in the individual, wherein, the administering delectably improves one Or more symptoms of MS hi the individual, and one or more therapeutic agents. In one embodiment, the therapeutic agent is a glucocorticoid. to specific embodiments, the ghicocorticoid b adrenocorticotropic hormone (ACTH), methy Iprednisotoite , or dexaroethasone. In another embodiment, the therapeutic agent is ah immimomodulatory or immtmosuppressive agent. In various specific embodiments, the immunomodulatory' or immunosuppressive agent is ΐΡΝβ- 1 a, IFN- 1 b, gliairiamer acetate, cyclophosphamide, methotrexate, azathioprine, cl*dribine, cyclosporine m mitoxmitrane. to other embodiments, the therapeutic agent is intravenous immunoglobulin, plasma exchange, Or sulfasalazine. In another embodiment, the individual is administered any combination of the foregoing therapeutic agents.
Treatment of Inflammatory Bowel Disease
1002441 to one embodiment, placental stem cells, placental stem cell populations, and/or compositions comprising placental stem cells or placental stem cell populations, are used to treat an individual bating, or at risk of developing, inflammatory bowel disease (IBD), e.g., Crohn’s disease or Ulcerative colitis. Thus, ih another aspect, provided hereto is a method of treating an individual having inflammatory bowel disease, or a symptom associated with inflammatory bowel disease, comprising administering to the individual a plurality of placental stem cells, or medium conditioned by placental stem cells, in an amount and for a time sufficient to delectably modulate, e.g., suppress an immune response to die individual. [0237] Crohn ¾ Disease, to one embodiment, the IBD is Crohn’s disease, sometimes referred to as ileitis or enteritis. Crohn’s disease is a chronic disorder tliat causes itffiammation of the digestive tract (also referred to as toe gastrointestinal, or 01, tract). Crohn’s disease can affect any part of toe 0l tract, firbm mouth to anus, but most comm only affects the lower part of the small intestine, referred to as the ileum. Five types of Crohn’s disease are known. Gastroduodenal Crohn’s disease afflicts toe stomach and duodenum (the faintest portionoffee «hall intestine). lejunoileitis is Crohn’s diseaseof the jejunum, the longest portion of the small intestine. Ileitis isCrohn's disease of the ileum, the lower portion of die small Intestine, Ileocolitis, die most common form ofCrohn'sdjsease, effects the ileum and colon. FinalIy, Crohn’s cotitis {Gfanulomatous colitis) affects the colon, and is distinguished from ulcerative colitis in that in Crohn’s colitis, them are often areas of healthy tissue between ureas tif diseased tissue, andCrohn¾cotiti$ can involve only the colon, without involving the rectum. Crohn’s disease is thought to arise from inappropriate reaction of the body’s irnmime system to antigens in the Gl tract, including, e,g., food, beneficial bacteria, etc., resulting man accumulation of white blood cells in the lining of the intestines.
Inflammation associated with Crohti’s disease has also been attributed to the action of fee cytokine tumor nebosis factor (TNF«a).
180245) Ulcerative colitis, In another embodiment, the IBD is ulcerative colitis. Ulcerative colitis is a disease that causes inflammation and sores (u!cers)in the lining of the rectum and/or colon. Ulcers fbim where inflammation has killed the cells that usually line the colon; the ulcers typically subsequently bleed and produce pus. When inflammation occurs in the rectum and lower part Of the colon, the disease ½ referred to as ulcerative proctitis. If the entire colon is affected, fee disease is called pancolttis. If only the left side of fee colon is affected, fee disease is referred to as limited or distal colitis : Symptoms of ulcerative colitis include, but are not limited to, abdominal paid, bloody diarrhea, fevers, nausea, abdominal cramps, anemia, fatigue, weight loss, loss Of appetite, rectal bleeding, loss of bodily fluids and nutrients, skin lesions, joint pain, and growth failure (in children). Ulcerative colitis can also cause complications stick as inflammation of the eye, liver disease, and osteoporosis. [0239] Thus, in one embodiment, provided herein is a method of treating an individual having an inflammatory' bowel disease, comprising administering a therapeutically effective amount of placental stem Cells to said individual, wherein said therapeutically effective amount is an amount feat results in a detectable improvement in at least one symptom of said inflammatory bowel disease (IBD). In a specific embodiment the IBD is Crohn’s disease. In a more specific embodiment, said Crohn’s disease is gastroduodenal Crohn's disease, jefenoileitis, ileitis, ileocolitis, or Crohn’S colitis. In another mote specific embodiment, said symptom is a symptom of Crohn’s disease» In a more specific embodiment, said symptom of Crohn’s disease is inflammation and swelling of a part of the GI tract, abdominal pain, frequent emptying of the bowel, and/or diarrhea. In another more specific embodiment» said symptom of Crohn’s disease is rectal bleeding, anemia, weight loss, arthritis, ikin problems, fever, toiCkening of the intestinal wall, formation of scar tissue in the intestines, formation of sores or ulcers in the intestine, development of cate or more fistulas in the intestinal wall , deve lopment of one or mote fissures in the antis, development of nutritional deficiencies (e.g.* deficiencies in one or more of proteins, calories, vitamins), development of kidney stones, dev elopment of gallstones, of diseases of the liver or biliary system. [0240] In another more specific etobodiuumt, the IBD ½ ulcerative colitis, to a motespecific embodiment, said Ulcerative colitis is Ulcerative proctitis, pancohtis, limited colitis or distal colitis, in another tome specific embodiment, said symptom is a symptom of ulcerative colitis: In a more specific embodiment, said symptom is abdominal pain, bloody diarrhea, fivers, nausea, abdominal cramps, anemia, fatigue, weight loss, loss of appetite, rectal bleeding, loss of bodily fluids and nutrients, Skin lesions, joint pain, and growth failure. In another more specific embodiment, the symptom is osteoporosis, eye inflammation, or liver disease.
160246] In another specific embodiment, said individual to whom placental stem cells are administered ¼ additionally administered one or more of a second therapy, wherein said second therapy comprises an anti-inflammatory agent, steroid, immune suppressor, and/or an antibiotic; Example? of anti-inflammatory drugs useful in the treatment of Crohn’s disease or ulcerative colitis include, but are not limited to, mesalamine, 5- ASA (5 -aminosalicylic acid) agents (e.g., ASACOIS (mesalamine, delayed-retease), PIPENTUM (Osalazine), PENTASA® (tnesalamme controlled-release)), sulfasalazine (a combination of 5-ASA and sutfapyridine), antiinflammatory antibodies (e.g. Infliximab (REM!CADES)), and the like. Examples of steroids useful to tihe treatment of Crohn’s disease or ulcerative colitis include, but are not limited to, cortisone, hydrocortisone, predisone, methylprednesone, and tile tike. Typically, as practiced ip die art - the dosage of steroid ts first delivered in a relatively large dose, followed by smaller dosages as inflammation subsides. Examples of immune suppressors useful in the treatment of Crohn’s disease include, but ate nor limited to, cyclosporine A, 6-mercaptopurine or azathioprine. Any antibiotic can be used ½ toe treatment of Crohn's disease, including, e.g, fKapiciltin* sulfonatnide. cephalospottim teiracyctine, and/or metronidazole EXAMPLES
EXAMPLE 1: METHODS OF REDUCING TF EXPRESSION / ACTIVITY
(00247 j Modulation of TF expression and/ or activity was assessed using various options.
First, treatment with various small molecules was assessed. As shown in Table I , small molecule treatment was not process compatible, resultirn-ng in reduced viability, and / or reduced nominal phenotype expression. table 1: Smalt molecule treatment.
Figure imgf000082_0001
00248 Other strategies, including antibody blocking, cell coating and genetic modification next were assessed; As shown in table 2, TF antibody blocking was not process compatible and cell coating significantly reduced viability and resulted in poor yield, Genetic mpdificatidii. proved $9 be the most successful arid effiCient method having no effect on viability or nominal panel expression and showing elective mductioti ra TF expression^,, TF active- clotting ¼nd coagulation. table 2: Alternative strategies for TK modulation
Figure imgf000084_0001
EXAMPLE lx CmSPR TISSUE FACTOR KXOCKOUT sgRNA/Cas9 RNP transfection
Table 3 TrueGuide** Synthetic sgRNAs for TF (M35533, ThermoFtsher Itmtrogen)
Figure imgf000085_0001
|O0249j At day 0» cells were seeded at indicated density in 6-well plate. At day I , mix
125ul of Opti-MEM medium^ 6.25ug of Cas9 nuclease (&A36493, ThermoFisherXnvitrogen), 1.2ug of sgRNA, and 12.5ul ofCas9 Plit$ reage«t (WCMAX00008, ThermoFtsher Invttrogen) in Tube t. Mixed I25nl of Opti-MEM tnedmm and 7.5ul of (#GM AX00008, ThermoFtsher invitrogen) in Tube 2. Immediately add solution from Tube! to Tube 2X then mix well, incubate complex for 5-10 mins at RT. Add 250ul of complex into each well. Incubate cells for 2 days at 37 ?C. After mcabatkm, remove APPL-001 culture medium and feed cells with fresh culture mediant;
TF activity assay (#ab 108906)
(002501 Prepare all reagents, working standards and samples. Standard Curve: Reconstitute the TF Standard with 0,8 ml reagent grade water to generate a solution of 250 pM Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the standard solution (250 pM) 1 :2 with Sample Dihient to FOduce 125, 62.5, 31.25, 15.63, 7.81 and 3.906 pM. Sample Diluent serves as the zero standard (0 pM). Freshly prepare the desired volume of Assay Mix by combining the following reagents. The values represent the volumes for one reaction: Assay Diluent 50 FV$ iOfoL, atiti PX ifofoL Add 7Q pLofthe Assay Mix to each well Add 10 μί> oftissue Factor Standard or sample to each well Mix gently. Incubate at 37°C for 30 minutes in a humid incubator to avoid drying out the wells. Add 20 pL ofFXa Substrate to each well and mix gently. Cover We¾ with a sealing tape and incubate at 3 ?*C and read absotbances oh a tnicroplate reader tit * wavelength of 405 ntn every 5 minutes ft» 35 minutes. Incubate micfbplate at 37°C after eachreading PGE2 potency assay
100251} Thawed APPL ceils Were plated and allowed to recover ½ growth medium, followed by conditioning in a medium containing the pro-inflammatory cytokine IL-Ιβ. During the conditioning period, APPL cells were stimulated by IL-1 ϋ to secrete POB2; After 24 hours, the CM was collected and cryopreserved at -80o€ until PGE2 analysis. The ELISA-based PGE2 analysis procedure follows foe protocol previously established by R&D in 2011 for Potency Assay Development ofPDA-OOl (Ran®, 2011). Initial development of the PDA-002 CM generation procedure was focused On the cell plating densities and the iise of lJW β as a stimulating agent. A range of cell plating densities (500 - 30,000 ceUs/cm2) Were evaluated in T- 75 flasks and the density Of 10,000 ceils/cra2 was incorporated into foe procedure. The incorporation ofIL-lp at lOng/mL during foe conditioning step was conftmted to be necessary to stimulate PGE2 secretion by PDA-002 cells. TtbE assay (002521 Genomic DN A was isolated from APPL using the Qiagen DNeasy® Blood & Tissue kit PCR reactions iwe earned out with 200 ng genomic DMA in according to manufacture instructions. PCR conditions were 1 min at 95°C (1 *), followed by 15 s at 95°C, 15 s at 55°€ and 1 min at 72°C (30*). The 40ul of PCR products were sent for sattger sequencing in Genewiz. Sequencing data were uploaded to https;//tide.nldn|/ for analysis.
The following primer pairs spanning the target site were used* hCD142EX3FI : TTC CTA TCC CTA CCC GOT TC hODl42EX3Rl: GTT ACCCTG ACA GAG AGC GT Resets
Part l ; Proof-ofconcept study: APPL 981, 974, 995 cell lines TF-gRNAs screening for high TFKO efficiency (602531 We tested four cell seeding densities at 4*105, 2x105, 1*165, Sx|64 ceHs/well in
6-well re flate for four pra-designedsgRNAsQnboth 981 and 995, FACS results showed that sgRNA B or C led to higher TFKO (>95%) than A or D (FIGS. 1A-IB), TIDE assay results were Consistent with FACS, showing high editing efhtiency of sgRNA B and C (FIGS. 1 A-1B),
FACS results also demonstrated that cell density had a ratearfcable effect on knockout efficiency. Among four seeding densities, 5*104 cells/welt gave out the highest TRJKO.· All these data suggested sgRNA B/C and seeding density at 5x104 cells/wetl was the optimal lipbfectioh condition.
Reproducibility of TFKO by Bpofection
(80254) To determine whether lipofectamiiMsmediared TFKO is reproducible, we transfected sgRNA B and C separately in multiple wells (n=4) in 981, 974 and 995 lines. FACS data showed that sgRNA-B or ~C delivered by lipofection ted td TFKO (>90%) in 981 and 974 and (>80%) in 995. TFKO by both sgRNAs was reproducible among the technical replicates (FIGS.2A4B).
TF activity reduction ¼ TFKO
1002551 TF triggers coagulation protease cascade and activates both FIX to FIXa and FX to FXa. This leads to the formation of low anmuntsof thrombin, which activates the cofactors FV and FVIll and cause the safety issues in patient treatment. Different MSC products display varying levels of TF, BM-MSC has low TF expression level: To comprehensively detennme TF activity tn APPL-TFKO lines, we tested it in both Whole cells and cell lysates. TF activity of APPL-TFKO is compared with control (non -edited) and human BM^MSd. In FIGS; 3A-3B, activity was reduced from control cells in both cell lines by >90%. Both whole cells and cell lysates showed remarkable reduction of TF activity; TF activi ty level in whole cells of APPL- TFKO was as low as that in BM-M SC.
Maintenance of TF express ion and activity in APPL-TFKO
100256) We generated APPL-TFKO at early passages (pO or pi ). These cells would be expanded to p6 as products. To determine whether TFKG was stably maintained in cell products at p6, we examined TF expression by FACS assay at each passage for 981 , 974 and 995. TF activity (whole cells) at p6 was measured by colorimetric assay, FIGS; 4A-4C demonstrated that both TFKO-B and TFKO-C wera maintained in 974 and 995 as cells expand (torn pi to p6. TFKO-B was stably maintained in .981 from pl to p4, however, not maintained at p5 mid p6. TP activityshowed that sgRNA^C mediated TFKO activity reduetionretained at p6, and it was as low as that in MSC, Observed increased relative TF activity of pd ceils were at both $81 and 974 atp6,
APPMTFKO proliferation f60257| We further investigated whether †FKQ affects the prolifetation of APPL, We documented imtial seeding ceil ntifnber and harvested cell number at each passage of 981, 974 and 995, That population doubting level (POL), which referred to die total number of times the cells in the population have· doubled since thetir primary isolation in vitro, were calculated- The accumulated PDLctirve showed that TFKO did not idter the proliferation of all three cell lines and both TFKO-B and TFKO-C demonstrated similar total PpL (FIG.5).
Nominal phenotype analysis of APPL-TFKQ
{602581 AFPLs were characterized by a panel ofmarkers,
CD lO-t-ZCDl 05+/CD20CH-/CD34". To characterize APPL-TFKO celt lines, we checked this nommal phencrfype markers by FACS for 981 , 974, and 995 lines. FIG; 6 showed that CD34 expression level is <1%, CDlO+ and CD 1054 level is >99%, and CD200 is >90% for all three cell lines. SgRNA*B/C did hot show difference in nominal genotype.
PGE2 secretion potency of APPL-TFKO
{602591 PGE2 is secreted by APPLs, suggesting as a potency indicator for APPL biological activities; We examined whether TFKO by sgRNA-B and -C would affect PGE2 secretion. APPL cells were stimulated with IL- lb. After 24 hours, secreted PGE2 was measured by ELISA. PGE2 secretion level Was normalized by XTT metabolic activity, PGE2 activity assay showed that 974 and 981 demonstrated similar PGE2 secretion profiles amongst the control and TFKO-B and -C. XTT Assay of metabolic activity showed similar results between control andKO-B with donor974 and 981 (FIG. 71- Part 2. Conformation Study: AE arid AL APPL-AE/AL-TFKO establishment
{60266} APPL-AE and -AL are standard APPL cell lines established ih CCT. In confirmation studies* we established APPL-AE and ~AL~TFKQ cdl lines with sgRNA-B. We examined IT expression level of these TFKO cell lines by FACS mid TIDE assay, to FACS, TFKO by gRNA-b achieves >95% in both standard cell lines (FIGS. 8A-8B),
TFKO confirmation by TIDE assay. IWtrn to further confirm TFKO in APPL eeU lines tested by FACS, we checked editing efficiency of by sanger sequencing and TIDE assay in 981, 97% AE and AL. Fig. 2.2. A is DNA gel imaging for product for senger sequencing, which cowed the editing region, FIG, 9 demonstrated ihatsgRN A*B led to a high editing efficiency (>85%) in ail four cell lines, which was consistent with FACS results. Editing efficiency is lower (~50%) by sgRNA-C m 974 cells With low R2 value dne to low quality Of FOR samples;
APPL-TFKO proliferation
{902621 We further investigated whether TFKO affects the proliferation of APPL standard cell lines. We documented initial seeding cell number and harvested cell number at each passage of AE and AL. Then population doubling level (FDL), which referred to the total number of times the cells in the population have doubled since their primary isolation in vitro, were calculated. In the fable, we listed Hie values of accumulated PDL and viability of cell harvesting at each passage for AE/AL-CTR and -ko B cells, The accumulated PDL curve showed that TFKO with sgRNA B did not alter the proliferation Of both AE and AL. TFKO did not affect the viability of AE and AL.
TF activity of four lines at late passage
|90263j TO compare the TF activity between APFL-CTR and APPL-ko B, we tested whole cells of 981, 974, AE, and AL at late passage, which were used in EAE animal studies. TF activity of APPL-TFKO is compared with control (non-edtted) and human BM^MSC. In FIG. II, TF activity of APPL-TFKO was reduced from control cells in both cell lines by >80% in 981, and >90% in 974, AE, and AL. The absolute values of TF activity demonstrated that TF activity Of all four lines with TFKO are close that of BM -MSC.
Nominal phenotype analysis of APPL-TFKO; AE and AL {60264| APPLs were characterized by a panel of markers,
CD!0F/Cp 165+/CD20tH-/GD34-. to characterize APPL-TFKO ccIl lines, we checked tMs nominal phenotype markets by FACS for AE and AL lines. FIG. 12 showed that CD34 expression level is <1%, CDJtft- and CD105+ level is >99%, and CD200 is >90% for both AE/AL-CTR and-ko B cells (FIO. |2)/TFKO-B did not alter nominal phenotype of AE and AL. BTR of APPtrTFKO: 4 lines
|00265) From previous research data, APPLs have been shown to possess immuntimodulatofy fimetions and APPLs suppress T-cell proliferation induced by CD3/CD28 stimulation, We investigated whether APPL-TFKQofd titles maintained suppression ofT-eeti proliferation by BTR assay. FIGS. I3A-13B showed that TFKO in 98 i < 974, AE and At cells inhibited boteCD4 and CDS T cell proliferation. The data suggested APPL-tFKO possess biplogicdfiyiettdns Of T-eeU proWfemtiott suppression.
Macrophage differentiation
(00266} Macrophages can be generated front monocytes in vitro arid undergo classicaiMI
(LPS^IFN-γ) or alternative M2 (IL-4) activation< Ptodnflammatory Mi macrophages protect against bacteria and viruses. M2 macrophages with antiinflammatory properties ate associated with wound healing and tissue repair4 Historical data suggested that APPL cells have immune regulatory properties including an anti-inflammatory activity, beneflting the wound heating process. In this study, we assessed the effect of APPL and APPL-TFKO cells on M1/M2 polarization. Fig. demonstrated that Control and TFKO cells increased the expression of M 1 marker CD206 and M2 marker CD80 in dOnor AL, 98 i , and 974, and increased CD8G only in donor AE. These data suggested that APPL-cOntrot and -TFKO induced polarization of macrophage to both M 1 and M2 phases.
Pari 3 ίη νίνο study: EAE animal model Cell preparation
(00267} To reach the target of 80 M cells fin EAE study, we thawed and expanded control tod TFKO of 98ϊψ2, 974-p2 to p4, and expanded control and TFKO of AE-p4, and AL-p4, to p5. Table 1 is the inventory of cells ready for in vivo study. We further mated post-thaw cell number and viability. All- of them had >85% post-tbaw viability. AE arid AL Were more than 80 M cells in post-thaw test
TF expression, IF activity and phenotype analysis
(00268) To ensure all these cells with corrected phenotypes, we tested tee TF expression,
TF activity and nominai phtootype panel markers. FACS results showed TFKO by sgRNA-8 significantly reduced TF expression of all four ceils ready for animal study FIG. 15). FIG. 15 demonstrated tee decreased TF activity in four APPL-TFKO cells compared!» control cells, which was consistent with TF expression data. Phenotype analysis showed all four TFKO lines maintains APPL phenotype (CD34-€D105+GD20(HCD10^).
(00269) PGE2 secretion potency of APPL-TFKO (002701 We have examined P6E2 potency of control and TFKO tit 981 and 974 lines. We further examined whether lTKOtiy sgRNA-B would affect PGE2 secretion in AE and AL, APPL cells were stimulated with IL- l b. After 24 hours, secreted PGE2 was measured by ELISA. PGE2 secretion level was normalized by XTT metabolic activity, PGE2 activity assay showed that AL demonstrated similar PGE2 secretion profiles between the control and TFKO-B. Afe-TP&O increased PGE2 secretion by 2 folds compared to control cells (FIG, 16),
Secretome analysis
(002711 APPL release extracellular vesicles and other soluble proteins or biologically active molecules, which collectively constitute, the MSC secretotne, in a paracrine manner; We investigated whether TFKO aEOc^pamcrine release Of secretome in 981,974, AE, and AL.
We profiled the cytokines and chemokines in collected cell culture media by both MILLTPLEX MAP Homan Cytokine/Chemokine Magnetic Bead Panel -Premixed 41 Plex and MSO.
Profiling data demonstrated no significant difference in between TFKO-B and control of ail 4 cell lines. Suggesting TFKO did not alter APPL secretome (FIG; 17).
½ vivo MOG BAB study
(90272) Next, we assessed safety of APPL-TFKO and APPL-control in vivo and evaluated their efficacy m EAE model. Both ApPL and APPL-TFKO smgle and repeat dose reduced BAB score and increased body weight compared to vehicle group. Particularly, APPL-TFKQ single dose gave out the lowest EAE score among these four group: (FIG. 19). These data suggested APPLrTFKO retained the efficacy in EAE in vivo model APPLrderived exosomes
(00273) The therapeutic effects of Mesenchymal stenv'stromal cells (MSCs) are largely mediated by paracrine factors including exosomes, which are a diameter of 40-150 nm membrane-bound vesicles with functions as mediators of cell-cell communication. MSC-derived exosomes contam cytokines, growth factors, bioactive lipids, RNAs, and proteinsS. Increasing evidence suggests that MSC-derived exosomes might represent a novel cell-free therapy with compelling advantages over parent MSCs such as no ride of tumor formation and lower immunogeuietty. We extracted exosomes from APPL cells and characterized the cytokines in APPL-derived exosomes. Table showed that cytokine, HGF* was highly detected in both APPL 981 control and TFKO cell lines; cytokines, including MCP-J , RANTES; and lL-8, had a low to moderate level. CRIPSR knockout off-tatget analysis by RNA-^ubnbittg
190274) CRISPR/Cas enables a targeted modification of DNA sequences. Despite then easeUnd efficient use, one limitation is the potential occurrence of associated off-target effects. Using the unbiased approach whole genome sequencing (WGS) is tiie moat common detection method allowing the identification of off-target effects in a less restricted way6. However, due to a high cost Of WGSt we fan a qttiOW and easier test, RNA-seqttencing, to analyze and compare the transcriptome of gene expression patterns in between APPL parental control cells and APPL- TFKO cells,
[00275} Three pairs of donors were tested* including AE, AL, and 981 ; We found the expression level for 3 genes only were consistently changed after TF gene was knocked out by CRIPS R/Cas9 in thesetbree donors (FIG. 26). Among these genes, 2 protein coding genes are wefi-clwacierized, mcludinglnterferon alpha-inducible protein 27 (JF127) and 2’-S*- OHgoadenylate Synthetase 2 (OAS2). IP127 were up-regulated by 2.35 folds in AL, 2.36 folds in AE, and 3.37 folds in 981 ; and OASi were up-regulated by 2.07 in AL, 2.96 in AE, and 3.42 in
981.
160276} IF127 is a hydrophobic mifocbdndrial protein composed of l22 amino acid [5).
Rosebeck and Leamanet at reported tliat 1EI27 maintains a low background expression in a variety ofmammalian cells and participates in multiple biological processes, including apoptosis and Congenital immunity. IFI27 expression was confirmed to be elevated m the psoriatic lesions and uterine fibroids, ovarian cancer, and other diseases, appearing to be a good marker for certain diseases (respiratory syncytial virus infection in preterm infants, nontnvasive biomarker in peripberal blood samples for immunoglobulin A nephropathy and membranous nephropathy, 190277} The OAS2 protein is a well-known innate immune activated antiviral enzyme catalyzing synthesis of 2 # -S' -oligoadenyiate for RNase Lactivation and inhibition of viral propagation. Overexpression of OAS2 has been reported in patients with inflammatory, autoimmune and malignant diseases, whereas its role in these conditions remains poorly understood. OAS2 expression Was upregulated m ail conditions and correlations between metby!ation and expression were seen in psoriasis and tongue SCO.
190278} TFKO in AL Were upregulated SCHiPl by 14.93 folds and Were downregulated
C$F3 by 50 folds and CXCL! by 10 folds. 190279} Sebwahnotnin interacting protein I (SCHIPI), Schipl interaction parmer Merlm belongs to die family of ERMproteins (Ezrin, Radix in and Moesin) that fonction not only as linkers between the cell membrane and the cortical cytoskeleton, but also as active modulators of the aetm eytoskeieton and membrane receptors tii the subcortical areas bf cellextensious.
Colony-stimulating foctor 3 (CSF3) is a cytokine used clinically tot promoting the production and release of hpiie maifow-derived hematopoietic stem cells and granulocyte [9], However, CSF3 has been shown to affect additional aspects of the innate and adaptive immune Systems. CSF3 inhibitioft increased T eeti infiltration, and decreased neoplasm devbfopment by 75% in die AOM/BSS model of CRC development [14]. These finding suggest potential pro* tumor effects of CSF3 signaling that are of garticufer concern because recombinant €$P3 ¼ currently used to prevent and treat febrile neutropenia secondary to chemotherapy . f0O281j C-X-C Motif Chdmokine Ligand 1 (CXCLl) acts as a chemoattractant for several immune cells, especially neutrophils. Diseases associated with CXCLl include ICaposi Sarcoma and Bacterial Meningitis. Among its related pathways are OCRS Pathway In Macrophages and PEDF Induced Signaling.
{00282} TFKO in AE were upregtiJated PCDHGB4 by 14475 folds, ACTAl by 333 folds, SNX22 by 46 folds, CD163 by 24.8 folds and were downregulated CIQTNF5 by 100 folds and AGAP5 by 30 folds.
100283} Protocadberin Gamma Subfamily B, 4 (PCDHGB4) belongs to the hugest mammalian Subgroup of the cadherin superfamily ofhomophilic cell-adhesion proteins. Diseases associated with PCDHGB4 include Papillary Craniopharyngioma.
100284} Actin Alpha 1 , Skeletal Muscle (ACTA 1 ) provides instructions for making a protein called skeletal alpha (a)-actrtu which is pari of the actin protein family. Skeletal ct-actin plays an important role in skeletal muscles Diseases associated with ACTAl include Myopathy, Scapuiohumeropcroneal and Ndmaline Myopathy 3. Among its related pathwaysare RhoGDi Pathway and PAK Pathway.
{00285} SNX22 (Sorting Mtutm 22). The protein encoded by this gene is a sorting nexin that is found in the cytoplasm, where it interacts with membrane-bound phosphatldylinoshol 3- phosphate. The encoded protein may play a role in intracellular trafficking. Diseases associated with SNX22 include Osteogenesis Imperfecta, Type lx and Hemochromatosis Type 2. (902861 duster of Diilerenttatiou $63 fC£>lB> Is foe high aBnity scavenger receptor for the hemoglobm-haptoglobin complex and in the absence of haptoglobin- with lower aBnityr for hemoglobin alone. It also is a marker of cells flroro the monocyte/macropltage lineage. CD 163 functions as innate immiine sensor ibr gram-positive and gram-negative bacteria. A soluble form of the receptor exists in plasma, and cerebrospinal fhhd. sCDl 63 is upregulated in a large range Of itiflaminatoty diseases including liver cirrhosis, type 2 diabetes, macrophage activation syndrome, Gaucher's disease, sepsis, Hi V infection, rbeumatoid artbritis and Hodgkin lymphoma. sCDI 63 is also upregulated id cerebrospinal fluid after subarachnoid Imemorrhage. (902871 Ciq and tumor necrosis factor related protein 5 (C1QTNF5) gene secreted and membrane-linked to a protein Which is strongly expressed in retinal pigment epithelium cells, A mutation in foe C1QTNF5 gene causes late-onsel retinal degeneration. More specifically, a single mtssense mutation (S163R) in foe encoded €1 QTNF5 protein cames foe Late-dnset retinal degeneration disease! L-ORD).
(002881 Arm AP With Gfpase Domain, Ankyriti Repeat And PH Domain 5 ( AGAP5) is related to GTPase activator activity. Among its related pathways are Endocytosts.
1602891 TFKO in 981 were upregulated FAM187A by 3553 folds, BIVM-ERCC5 by 22 folds, and were down-regulated TMEFF1 by 5000 folds, EXOSC6 by 25 folds, and NPB by 1 $ folds.
(002901 Family With Sequence Similarity 187 Member A (FAM187A) is related to protein homodimerization activity. Diseases associated with FAM187A include Ciliary Dyskinesia, Primary, $7 and Chromosome 17Q12 Duplication Syndrome.
(002911 B1VM-ERCC5 is related tp single-stranded DNA binding andnuclease activity. Diseases associated with BIVM-ERCC5 include Cerebrooculofocfoskeletal Syndrome 3 and Xeroderma Pigmentosum, Comple
Figure imgf000094_0001
Group G. Among its related pathways are Nucleotide excision repair.
(00292) TMEFFl is a novel transmembr ane protein, composed of two follisatin domains and a unique epidermal growth factor (EGF)-like region. These structural domains suggest a role for TMEFFl in tbe regulation of growth factor signaling either as a ligand precursor , a membrane-bound receptor or as a binding protein for growth factors, TMEFFl may inhibit NODAL and BMP signaling duringneural patterning and may be a tutnotsuppressor in brafo cancers. (602931 ExoSonte complex exonuclease Mills (EXOSCO) constitutes one of the subunits of the multisubunit particle called tile exosome complex, which mediates mRN A degradation. Disease® associated with EXOSC6 include X- Linked Nephrolithiasis Type 1 and Planter Wart* (602941 Neuropeptide B (NPB) may regulate feeding, pain perception, and stress in rodents, Diseases associated with Nf*R include Niemann-Pick Disease, Type B sud Gallbiadder Papillomatosis.
References
1. James Edinger, et al Placenta-Derived Mesenchymal Stromal Cells: Modulation of immunity and Inflammation.
2. Denis Nt Silachev, et al Effect of MSG» and MSC-Derived Extracellular Vesicles on Human Blood Coagulation. Cells. 2619 Mar; 8(3): 258.
3. Barbara A Christy, et al. PirocoagUtant activity of human mesenchymal stem cells. J Trauma Acute Care Surg.2017 Jul;83(l Suppl t):Sl64-S169.
4. Klaus Ley. Ml Means Kill; M2 Means HeaU Immunol October t, 2017, 199(7) 2191-
2193.
5. Kan Yin, el at Exosomes from mesenchymal stan/stromal belli: a new therapeutic paradigm. Bitimarfcer Research volume 7, Article number: 8 (2019).
6. Dominik Modrzejewski, et al. What ½ the available evidence for die range of applications of genome-editing as a new tool for plant trait modification and the potential occurrence of associatedofTtarget effects: a systematic map. Environmental Evidence volume 8, Article number: 27 (2019),
Equivalents:
(002951 the present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to ½It within the scope of the appended claims.
(6629$! Various publications, patents and patent applications are cited herein^ the disclosures of which arc incorporated by reference m their entireties.

Claims

WHAT IS CLAIMED:
1. A: population of placental stem cells which, are CDlOV, 0334», CD I d$4, CD2CM>K and CD 142
2. The population of placenta! stem cells Of claim l which are additionally negative for one or more markers selected from the group consisting of CDl 17, CD45, and CD133.
3.. The population, of placental stem ceils of claim 2, which are additionally negative for
CD 117,
4. The population of placenta ! stem cells of claim. 2., which am addi ti onally negati ve lor CD45,
5. The population. Of placental stem cells of claim 2, which are additionally negati ve for CD 133.
6.: The population of placen tal stem cells of any one of claims tfoy which am additionally positive for one or more markets selected from the group consisting of CD44, CP90, 0et-4, and ELA-G,
7· The population, of placental stem cells of claim. 6, which am additionally positive for 0344,
8. The population^ of ipbventai stem cells of claim 6, winch are additionally positive for CD9CI.
9. The population of placental stem: cells of claim 6, which am additional iy positive for Oci-
4.
10. The population of placental stem cells of claim. 6, which am additionally posi tive for Ei,A-G:
11. The population of placenta] stem cells of any one of claims i- 10:. wherein the cells are genetically modified.
12. The population: of placental stem cells of claim i O, wherein the genetic modification is a tissue ihetor (CD 142, F3 gene) knockout.
13. Tfoe population of placental stem cells of claim 14 wherein the genetic modificatiod i$ a CRISPtt tissue Sew <CD142, F3 gene) knockout.
14 The population of placetital stem cells of any one of claims M3, wherein the cells induce increased anti-inflamatory maeropbageactmty relative to CD142†PDAC colls,
15. The population of placental stem cells Of claim H, wherein the uacreased anti- mflammatoty activity comprises increased expression of €D80 on M2 macrophages.
16; The population of placental stem cells of any one of claims M3, wherein the cells induce increased pro-mBammatoiy macrophage activity relative to CD142T PDAC cell^
17. Thepopuiation of placental stem cells of claim 16, wherein the increased anti- inflammatory activity comprises increased expression of CD206 cm Ml macrophages.
18. The population of placental stem cells of any one of claims 1-17, wherein the cells have increased anti-inflammatory properties relative to CD142+ PDAC cells.
19. T¾e population of placental stmt cells of any one of claims 1-18 , wher ei n the cells express increased an increased marker of activity under inflammatory conditions relative to CD142+ PDAC cells.
20. The population of placental stem cells of claim 1% wherein the increased marker of activity ¾ P0E2,
21. The population of placental stem cells of claim 19 or claim 24 wherein the inflammatory condition is IL- tb stimiilation,
22. A pbannacueutical composition comprisingthe population of placemal stem cells of any ohe of claims 1-21 and a pharmaceutically acceptable carrier.
23. A method of neating a disease or condition ½ a subject ½ need thereof, the method comprising tiie step of administering to the subject a therapeutically effective amount of the population of placental stem cells of any one of claims Ϊ-21 or the pharmaceutical composition of claim 22, so as to therein treat the subject.
24. The method of claim 23, wherein the subject is amammaL
25. The method of claim 24, wherein the subject is a human.
26. The method of any one of claims 23-25, wherein die administration is intravenous,
27. The method of any one of claims 23-25, wherein the administration is local.
28. The method of any one of claims 23-25, wherein the administration is intramuscular.
29. The method of any one of claims 23-25, wherein the subject has an autoimmune disease.
30. The method of claim 29, wherein the autoimmune disease is multiple sclerosis.
31. The method of any ohe of claims 23-25, wherein die subject has aninftammatoty bowel disease.
32. The method of claim 31 , Wherein tbe inflaramatoiy bowel disease is Crohn’s disease.
33. A method of suppressing an immune response comprising contacting a plurality of immune cells with a population of placental stem cells of any one of claims 1 -21 for a tithe sufficient for said placental stem cells to delectably suppress an iimmme response.
34. The method of claim 33, whereto said plurality of immune cells are T cells or NK (natural killer) cells.
35. The method Of claim 34, wherein said T cells are CD4+ T cells.
36. Tbe method of claim 34, wherein said T cells ate CD84 T cells.
37. Tbe method of claim 33, wherein slid con tacting is performed in vitro.
38. Tbe method of claim 33, wherem said contacting is performed m viva,
39. The method of claim 38, wherein said contacting is performed in a mammalian subject.
40. The method of claim 39, wherein said mammalian subject ½ a human subject
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